s2fieldsofstudy: Biology Medicine

text:
Toll-like receptor 2-mediated NF-kappa B pathway activation in ocular surface epithelial cells

Background Gram-positive bacteria stimulate Toll-like receptor (TLR) 2 and then activate the pro-inflammatory nuclear factor-kappa B (NF-κB) pathway. As the human ocular surface is heavily colonised by gram-positive cocci bacteria, a balance of activation/repression of NF-κB target genes is essential to avoid uncontrolled infection or autoimmune-related inflammation. It is advantageous to test NF-κB targeting molecules in an ocular surface culture system that allows assessment of temporal NF-κB activation in a longitudinal fashion without destruction of cells. Such initial testing under standardised conditions should reduce the number of molecules that progress to further evaluation in animal models. This study aims to establish an in-vitro cell culture system to assess NF-κB activation in the context of ocular surface cells. Methods NF-κB activity was evaluated through a secretory alkaline phosphatase reporter assay (SEAP). Immunoblots and immunofluorescence were used to examine IκBα phosphorylation and p65/p50 nuclear localization. Monocyte chemoattractant protein-1 (MCP-1) transcripts were evaluated by real time PCR and protein levels were measured by ELISA. Results NF-κB activity in HCE-T cells treated with TLR2 activator Pam3CSK4 was higher than control cells at both 6 and 24 h. Pam3CSK4-stimulated NF-κB activation was inhibited by IκK inhibitors, Wedelolactone and BMS-345541. In Pam3CSK4 treated cells, active NF-κB subunits p50 and p65 increased in cell nuclear fractions as early as 1.5 h. Although the level of total IκB-α remained constant, phospho-IκB-α increased with treatment over time. In the culture media of Pam3CSK4-stimulated cells, MCP-1 protein level was increased, which was suppressed in the presence of IκK inhibitors. Conclusion NF-κB pathway can be activated by the TLR2 ligand and inhibited by IκK inhibitors in the ocular surface cell culture system. This cell culture system may be used to evaluate TLR-related innate defences in ocular surface diseases.

Background
The ocular surface is constantly exposed to the colonization of various commensal microbes, playing an important role in defence against microbes and other inflammatory insults [1,2]. The nuclear factor kappa (NFκ) B transcription pathway is the central regulator of ocular surface inflammation and disease [3][4][5][6][7]. Toll-like receptors (TLRs) are a family of transmembrane receptors that recognize microbial pathogens and trigger early innate immune responses leading to inflammation in mammalian cells [8][9][10]. TLRs present on the membrane of ocular surface cells can recognize and bind to a variety of microbial components including bacterial lipopeptides, lipopolysaccharide (LPS), flagellin, viral dsRNA, ssRNA and other ligands [11][12][13]. On activation of TLR, subsequent signalling of the cells can activate the master kinase called the Ikappa-B kinase (IκK), which phosphorylates the Ikappa-B alpha (IκBα) subunit of NF-κB. The phosphorylated IκBα undergoes degradation, releasing the p50 and p65 subunits of NF-κB, which translocate to the cellular nuclei to bind to the promoter of target genes [3,8,14]. Through upregulation, and less often, down-regulation of target genes, this signalling pathway has a major influence on the local immune defence and may be critical in control of ocular infections.
The regulation of NF-κB is highly context-and tissuedependent, so it is important to define the triggers and targets of this pathway in healthy ocular surface and in disease. TLR2 is reported to function as a Gram-positive bacteria sensor in the cornea [12]. In Gram-positive bacterial infections, pro-inflammatory mediators such as TNF-alpha, interleukin (IL)-6, IL-8 and the adhesion molecule ICAM-1 were secreted by human cornea epithelial cells through activation of NF-κB [15][16][17]. The propagation of inflammation by these molecules can increase the severity of infection. On the other hand, NF-κB-mediated expression of anti-microbial molecules human defensin (hBD)-2 and homeostatic molecules manganese superoxide dismutase may help to control the pathogenicity of microbes or increase defences against oxidative stress [15,18]. It is therefore important to understand how NF-κB is finely tuned in health and disease involving ocular surface cells, so that appropriate therapeutic strategies can be implemented to protect the eye against ocular infections [8].
We aim to describe the activation of NF-κB in an invitro system of ocular surface cells using promoter assay and other approaches, induced by the presence of a TLR2 ligand.

Cells
Human SV-40 immortalized corneal epithelial cell line (HCE-T) cell line was obtained from the Riken Cell Bank (RCB2280) [19]. Cells were cultured with DMEM/F-12 (Life Technologies, CA, USA) medium supplemented with 5% foetal bovine serum (FBS) (Life Technologies, CA, USA) and maintained in a 37°C incubator with 5% CO 2 . Medium was changed every 2 days. Cells with passage no. 69-73 were used in this study.

Transfection, IκK inhibitor treatment and Pam3CSK4 stimulation
HCE-T cells were cultured to 90% confluent in 6-well plates and changed to fresh medium before transfection. Six microliters of Fugene 6 Transfection Reagent (Promega, Madison, USA) and 200 μl of Epilife medium (Life Technologies, CA, USA) were mixed and incubated at room temperature for 5 min. Subsequently, vectors (2 μg) pSEAP-basic or pSEAP-NF-κB (Clontech Laboratories, Mountain View, CA) were added to the mixture and incubated at room temperature for 15 min. The mixture was then added to the cells and incubated in a 37°C incubator with 5% CO 2 overnight.
IκK inhibitor Wedelolactone (Calbiochem, San Diego, CA) or BMS-345541 (Calbiochem, San Diego, CA) was added to select wells, which were transfected with pSEAP-NF-κB at a concentration of 10 μM or 5 μM, respectively. Pam3CSK4 is a well-established TLR2 specific ligand [20]. Depending on the experiment group assignment, Pam3CSK4 was added to selected wells at a final concentration of 250 ng/mL an hour later. The assignment of cell transfection and treatment groups is depicted in Fig. 1a.
NF-κB transcriptional activity detected by secreted alkaline phosphatase (SEAP) assay NF-κB transcriptional activity was detected using the Great EscAPe SEAP detection kit (Clontech Laboratories, Mountain View, CA) following the manufacturer's instruction. Briefly, culture media (400 μl) were collected from cells transfected with different pSEAP vectors with or without Pam3CSK4 stimulation and IκK inhibitors treatment. The culture media were centrifuged at 12,000 rpm for 10 s before adding to a 96-well black walled plate (12 μl per well). Fresh media was added (48 μl per well) to the culture medium. Serial dilutions of the positive control alkaline phosphatase were also added to the same 96-well plate. The plate was sealed with adhesive aluminium foil, incubated at 65°C for 30 min, and then cooled on ice for 2-3 min. After equilibrium to room temperature, 60 μl of assay buffer was added to each well and the plate was incubated for 5 min at room temperature. Subsequently, 60 μl of substrate working solution was added and the plate was incubated at room temperature for 10 min. Chemiluminescence was measured by Tecan GENios (Tecan, Männedorf, Switzerland).

Nuclear extraction
Nuclear extraction from HCE-T cells stimulated with Pam3CSK4 was performed using the nuclear extraction kit (Active Motif, Carlsbad, CA) following the manufacturer's instruction. Briefly, HCE-T cells were stimulated with Pam3CSK4 (250 ng/ml) for 0, 1, 1.5 and 2 h in 100 mm culture plates, washed with 10 ml of cold PBS, and scrapped off from culture plates with 400 μl of cold PBS containing protease and phosphatase inhibitors (Roche Applied Science, Penzburg, Germany). Scrapped cells were transferred to pre-chilled tubes and centrifuged for 5 min at 500 rpm at 4°C. Cell pellet was re-suspended in 500 μl of 1X hypotonic buffer and incubated on ice for 15 min. Twenty-five microliters of detergent was added to the cell pellet suspension and centrifuged for 30 s at 14,000 rpm at 4°C. The pellet was re-suspended in 50 μl of complete lysis buffer and incubated for 30 min on ice with shaking at 150 rpm, and then centrifuged at 14,000 rpm for 10 min at 4°C. The nuclear containing supernatant was transferred into pre-chilled 1.5 ml tubes and stored at −80°C until use. Protein concentration was determined by BCA method following manufacturer's instruction [21].

Western Blot
Western blots were performed as described previously [22]. Briefly, protein samples (30 μg per lane) were separated in 12% SDS-PAGE gel and transferred to PVDF membranes. The membranes were then blocked with 5% BSA in TBS-Tween 20 (TBS-T) for 1 h at room temperature and incubated with primary antibodies for 2 h at room temperature. After that, the membranes were incubated with horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature, washed with TBS-T for three times (5 min each time), incubated with SuperSignal West Pico chemiluminescent substrates (Pierce Biotechnology, Rockford, USA) and signals were visualized on X-ray films. Primary and secondary antibodies used in this study are listed in Table 1.

Immunofluorescence staining
Immunofluorescent staining of nuclear translocation of NF-κB active subunits, p50 and p65, were performed according to a previous protocol [23]. In summary, cells seeded in chamber slides treated with or without Pam3CSK4 were fixed with 100% methanol for 10 min at room temperature, washed with PBS, permeated in PBS containing 0.15% Triton X-100 for 15 min, blocked with 4% BSA in PBS containing 0.1% Triton X-100 (Sigma) for 1 h, then incubated with primary antibodies at 4°C overnight. After washing with PBS containing 0.1% Tween-20, cells were incubated with Alexa Fluor 488-conjugated secondary antibodies at room temperature for 45 min. Subsequently, chamber walls were removed and slides were mounted with VectaShield mounting medium with DAPI (Vector Lab, Burlingame, USA). Slides were observed and imaged using a Zeiss Axioplan 2 fluorescence microscope (Zeiss, Oberkochen, Germany). Primary and secondary antibodies used are listed in Table 1.

Enzyme-linked immunosorbent assay (ELISA)
MCP-1 protein in the culture media was quantified by ELISA (R&D system) following the manufacturer's instruction. Briefly, 200 μl of standards and culture medium were added to microplates coated with MCP-1 antibody and incubated for 2 h at room temperature. Solution from each well was aspirated and 400 μl of Fig. 1 a NF-κB transcriptional activity assay. HCET cells were transfected with pSEAP-basic or pSEAP-NF-κB vector, treated with Pam3CSK4 (a TLR2 ligand) for 6 and 24 h with or without the presence of IκK inhibitors Wedelolactone and BMS-345541. Height of each bar represents mean of two independent experiments and is normalized to negative control (pSEAP-basic). The error bars represent standard error of the means. b HCE-T cells were treated with Pam3CSK4 for various time intervals and nuclear fraction of treated cells were immunoblotted with antibodies against p50 and p65. c HCE-T cells seeded in chamber slides were treated with Pam3CSK4 for 4 h and immunofluorescent staining was performed using primary antibodies against p65 and p50, and Alexa Fluor 488 conjugated secondary antibody (green). Representative images are shown. Scale bar = 50 μm. d. HCE-T cells were treated with Pam3CSK4 with or without IκK inhibitors for various time intervals and total cell lysate was immunoblotted with antibodies against phospho-IκB-α and total IκB-α. GAPDH was used as the endogenous loading control. *: P < 0.05 washing buffer added. The wash step was repeated two more times. MCP-1 conjugates (200 μl) were added to each well and incubated for 1 h at room temperature. Subsequently, the microplates were washed three times with washing buffer, incubated with substrate solution (200 μl/well) for 30 min followed by the stop solution (50 μl/well). The optical density of each well was determined by Tecan GENios Pro microplate reader at 450 nm. Actual MCP-1 concentrations were determined using the standard curve generated with MCP-1 standards of known concentrations.

Real time quantification PCR
Real time PCR was performed as described previously according to the manufacturer's instructions [24]. In summary, total RNA was extracted from HCE-T cells using the RNeasy Mini Kit (Qiagen, Hilden Germany) and RNA concentration determined by the nanodrop method [25]. One microgram of RNA for each condition was used to synthesize cDNA. First strand synthesis was performed using Superscript II Reverse Transcriptase (Life Technologies, CA, USA) and real time PCR reaction was performed with Roche UPL Mastermix and an appropriate probe from the human library (Roche Applied Science, Penzburg, Germany). Forward and reverse primers used are: 5'TTCTGTGCCTGCTGCTCAT3' and 5'GGGGCATTG ATTGCATCT3' respectively. GAPDH was used as the endogenous control, forward and reverse primers are 5' AGCCACATCGCTGAGACA3' and 5'GCCCAATACG ACCAAATCC3' respectively. PCR cycles were performed on Roche LightCycler 480 (Roche Diagnostics, Basel, Switzerland) with the following conditions: denaturation at 95°C for 10 min, followed by 24 cycles of denaturation at 95°C for 10 s, annealing at 54°C for 10 s and extension at 72°C for 30 s. Delta-Delta Ct method was used to analyse data and fold change was expressed relative to GAPDH levels.

Statistical analysis
In the NF-кB activity assay, we performed the cell cultures in 3 independent wells and repeated 3 further wells on a different occasion. From each of the wells, the culture media were removed and split into 4 replicates for evaluation of the luminescence. The mean of the 4 replicates (which originated from a single well of cells) was evaluated. The means were used to calculate overall mean of the wells that had the same experimental treatment. To demonstrate overall findings in a graphical format, the means over the 2 occasions (which had 6 independent wells) were then calculated. The same calculation method was used for MCP-1 real time PCR and ELISA assay. All results are expressed as the mean ± standard error of the mean. Statistical analyses were performed using student's t-test for statistical comparison between different conditions. P values less than 0.05 were considered statistically significant.

NF-κB was activated through TLR2 in human corneal epithelial cells
The NF-κB activity of HCE-T cells stimulated with Pam3CSK4 for 6 and 24 h was compared to the unstimulated cells using SEAP assay. At both 6 and 24 h, Pam3CSK4 treated HCE-T cells showed significantly higher levels of NF-κB activity compared to controls (Fig. 1a, column 3 compared to columns 1 and 2). Protein level of NF-κB active subunit p50 increased in the nuclear fractions of Pam3CSK4 treated cells as early as 1.5 h (Fig. 1b). At the same time, cytosolic p50 and p65 decreased over time (data not shown). Immunostaining of the HCE-T cells treated with Pam3CSK4 over the time intervals 0, 1, 2 and 4 h showed increase of p50 and p65 in the nuclei (data for 4 h shown in Fig. 1c). There was an increase of phospho-IκB-α overtime with Pam3CSK4 treatment, while the level of total IκB-α protein remained relatively constant (Fig. 1d).

MCP-1 was up-regulated by TLR2-NF-κB activation
MCP-1 also known as chemokine (C-C motif ) ligand 2 (CCL2) is one of the critical regulators for leukocyte recruitment during cornea infection [26,27]. Transcript levels of MCP-1 were evaluated by qPCR in Pam3CSK4 treated and un-treated cells. Consistent with the NF-κB activity SEAP assay, at both 6 and 24 h, Pam3CSK4treated HCE-T cells showed higher levels of MCP-1 compared to controls (Fig. 2a, column 3 compared to columns 1 and 2). MCP-1 up-regulation was similarly inhibited with IκK inhibitors Wedelolactone and BMS-345541 (Fig. 2a, column 3 compared to columns 5 and 7). ELISA was used to examine MCP-1 protein levels in the culture media. Results showed that after 24 h treatment, MCP-1 protein in the medium was also up-regulated after Pam3CSK4 stimulation and it was suppressible with BMS-345541 (Fig. 2b) as in the case of the transcript levels.

Discussion
In this study, we found that in HCE-T cells, NF-κB activity was increased with TLR2 ligand Pam3CSK4 treatment, and the phosphorylation level of IκB-α was also increased with the addition of Pam3CSK4, while total IκB-α protein level remained the same. NF-κB active subunit p50 translocated to the cell nuclei as early as 1.5 h after Pam3CSK4 treatment; NF-κB activation was mediated by IκK. The chemokine MCP-1 was upregulated by the TLR2 NF-κB pathway at both the transcript and protein levels, and this was also mediated by IκK. Four previous studies evaluated the activation of NF-κB downstream of TLR-2 in cornea epithelial cells.
One study investigated NF-κB using the detection of p65 in the nuclear fractions of cells [16], another study investigated NF-κB activity in cell lysate with anti-NF-κB antibodies, whereas two other studies examined the levels of pIκB-α [15,17]. Neither of these examined the expression of MCP-1 as a possible target, and in addition, the previous studies in corneal epithelial cells did not employ NF-κB promoter assays. In our current report, we examined the activation of NF-κB using a fine tune promoter SEAP assay and reported the regulation of MCP-1 through TLR2 NF-κB pathway. The advantage of using SEAP assay is that the culture medium can be analysed at more than one time point in the experiment without destroying the cells. Two studies have shown apparently contradictory results when using peptidoglycan (PGN) to treat human corneal epithelial cells, one group found activation of NF-κB as well as increases in IL-6 and IL-8 expressions  [17], but not the other [28]. The controversy in this area cannot be easily resolved unless researchers agree to use a common testing strategy for screening potential drugs. We hope that the methodology we propose will be adopted by others and this should help to prevent confusing results from such studies. The strength of this study is the use of more than one approach to investigate the TLR2-NF-κB pathway. The limitation of the study is that in-vivo animal models of Gram-positive infection was not investigated and compared to our in-vitro findings.
In many experimental scenarios, it is valuable to screen a variety of potential new therapeutic agents that may treat Gram-positive eye infections. It is too expensive and resource intensive to investigate all these agents in animal studies in the first instance. It is therefore advantageous to be able to rapidly investigate NF-κB agents, especially those suspected to act via the IκK kinases, in a cell culture system such as the one in our current study to shortlist more promising candidates and reduce the number of molecules that progress to further evaluation in animal models.

Conclusions
In conclusion, the NF-κB pathway can be activated by the TLR2 ligand and inhibited by IκK inhibitors in the ocular surface cell culture system. This cell culture system may be used to evaluate TLR-related innate defences in ocular surface diseases.


extfieldsofstudy: Medicine Biology

text:
Virus inactivation in stored human urine, sludge and animal manure under typical conditions of storage or mesophilic anaerobic digestion†

Viruses represent major disease transmitting agents carried by human excreta and animal manure. Understanding virus inactivation is therefore essential in preventing microbial spread due to inadequate treatment of these materials. Here, we investigated the inactivation kinetics of the single-stranded (ss) RNA phage MS2, DNA phages T4 and ΦX174, andthe double-stranded DNA human adenovirus in stored human urine, sludge, and animal manure, at temperatures and pH valuestypical of storage under naturally occurring conditions or mesophilic anaerobic digestion (<40 °C). The ssRNA phage MS2 was most readily inactivated in all samples compared to the other viruses tested. This is consistent with previous findings in wellcontrolled buffer solutions of similar composition, where inactivation was found to be governedby bases (NH3, carbonate, hydroxide) that catalyze the transesterification and cleavage of the ssRNA. Correspondingly, MS2 inactivation kinetics in real matrices could be adequately modelled by only taking into account the effects of temperature, pH, carbonate and ammonia on the integrity of ssRNA. DNA viruses were more persistent compared to MS2;however, inactivation in selected sludge and manure samples proceeded at faster rates compared to well-controlled buffersolutions of similar composition. This indicates a contribution of microbial or enzymatic activity to inactivation of DNA viruses. Overall, this study identifies the most important factors contributing to inactivation of viruses in human excreta and manure, and highlights the differences in inactivation kinetics and mechanisms between ssRNA and DNA viruses.

Introduction
Prior to reuse or discharge, human excreta and animal manure (HEAM) need to be correctly managed to avoid the transmission of pollutants (macro-and micro-pollutants) and pathogens to the environment. [1][2][3][4] If adequately treated, however, environmental pollution and public health issues resulting from the disposal of HEAM can be minimized. [5][6][7][8] Organized excreta containment, collection and treatment is one of the pillars in breaking the transmission cycle of fecalorally transmitted pathogens. 9 Currently only a small percent-age of human excreta collected is safely managed, in particular in cities in lower-income countries, though on-site storage or treatment offer an opportunity to reduce the pathogen load before disposal. 10,11 On-site treatment should in particular focus on viruses and helminths, since they were shown to be the most persistent pathogens in excreta, in particular during storage at ambient temperature. [12][13][14] Their inactivation kinetics, and the factors that promote inactivation in HEAM, however, remain poorly understood.
In previous work, we characterized virus inactivation in well-controlled, homogenous laboratory solutions under conditions of pH, temperature and chemical composition typically encountered during storage or mesophilic anaerobic digestion of HEAM. 15,16 Single-stranded (ss)RNA viruses, such as enteroviruses or MS2 coliphage, were shown to be rapidly inactivated. Inactivation resulted from base-catalyzed transesterification of the ssRNA, which causes the genome to cleave. 15 The efficiency of a base to induce inactivation depends on the pK a of its conjugated acid and its concentration. Under the solution conditions considered in Decrey et al., 15 the most important bases promoting inactivation were hydroxide and ammonia, though other bases such as (bi-)carbonate also contributed. Based on this mechanistic insight, a model to estimate the inactivation rate constant under typical urine, sludge and manure storage conditions was established for MS2 coliphage, which was shown to be a conservative surrogate of other ssRNA viruses in such conditions. 16 Using the solution composition as the input, this model was able to accurately estimate the MS2 inactivation rate constant at 35°C and over a pH range of 7.5-9.5. In contrast to ssRNA viruses, double-stranded (ds) RNA and both ssDNA and dsDNA viruses, which are not amenable to genome transesterification, exhibited low inactivation rates under the same conditions. More extreme conditions of pH or temperature, such as those encountered in thermophilic digestion or alkaline treatment, were required to achieve appreciable inactivation rates. 16 Compared to laboratory solutions, real HEAM matrices exhibit a higher level of complexity: they contain particles, additional chemical constituents (e.g., metals or organic acids), as well as live communities of microorganisms that may contribute to virus inactivation. The current study extends our understanding of virus inactivation from laboratory solutions to real matrices associated with on-site HEAM storage (sludge, stored human urine, animal manure). Specifically, we aim to determine if the principles of virus inactivation established in well-controlled solution also apply to HEAM matrices. Our objectives were i) to validate the previous approach to estimate MS2 inactivation for the more complex conditions encountered in HEAM; and ii) to establish if the inactivation trends observed in laboratory solutions for viruses with different genome types correspond to those in real matrices. To attain these objectives, the pH, temperature and ion composition of different (diluted) stored urine, sludge and manure solutions was determined, and MS2 inactivation in these solutions was monitored and compared to the predicted inactivation. Furthermore, the inactivation kinetics of the base-sensitive ssRNA phage MS2 as well as the more resistant dsDNA phage T4, ssDNA phage ΦX174 and the dsDNA human adenovirus (HAdV) were determined in stored urine, sludge and manure, and were compared to results from controlled laboratory studies. The phages were chosen to span a range of susceptibilities to solution conditions typical of HEAM, and to represent different genome types. 16 HAdV was chosen as a representative of a base-resistant human virus. Finally, experiments were conducted to determine the effect of HEAM-associated environmental parameters, such as microbial activity or the presence of metals, on inactivation.

Virus and cells
HAdV type 2 was provided by Rosina Gironès (Laboratory of Virus Contaminants of Water and Food, University of Barce-lona). HAdV was propagated on A549 human lung carcinoma epithelial cells, provided by the University Hospital of Lausanne. A549 cells were cultivated in high-glucose, pyruvate Dulbecco's modified Eagle's medium (DMEM; Invitrogen). The media was supplemented with penicillin (20 U mL −1 ), streptomycin (20 μg mL −1 ) (Invitrogen), and 2 or 10% fetal bovine serum (FBS; Invitrogen) and cells were incubated at 37°C in 5% CO 2 and 95% humidity. Viruses were propagated by spiking 10 μl of HAdV (10 10 -10 11 most probable number of cytopathogenic units (MPNCU) mL −1 ) into 160 cm 2 flasks (TPP Techno Plastic Products, Trasadingen, Switzerland) containing 95% confluent cells, and were purified as described in Bosshard et al. 17 From each flask, 1 ml of samples containing 10 10 -10 11 MPNCU mL −1 of HAdV was collected and stored at 4°C as virus stocks for the experiments. New stocks were produced before each set of experiments. Virus titers were determined as MPNCU mL −1 from 5 × 100 μl of samples on 96-wells plates (Greiner Bio-One, Frickenhausen, Germany) as described in Bosshard et al. 17 Briefly, the DMEM containing 10% FBS on a 95% confluent cell monolayer was replaced by 100 μl of virus solution and augmented with 200 μl of DMEM containing 2% FBS. Cytopathogenic units could be discerned after an incubation time of 14 days. The detection limit was 10 2 -10 3 MPNCU mL −1 .

Phages and bacteria
Coliphages MS2 (DSMZ 13767) and ΦX174 (DSMZ 4497) and their host Escherichia coli (DSMZ 5695 and DSMZ 13127, respectively) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Coliphage T4 and its E. coli host (B1) were provided by Petr Leiman (Laboratory of Structural Biology and Biophysics, EPFL). Media used to grow E. coli B1 were free of any antibiotics. All phages were propagated and purified as described previously. 18 Stock solutions were stored in the fridge, and the same stocks were used for all the experiments. Infectivity was assessed using the double agar layer method as described elsewhere. 19 The detection limit for all phages was 300 PFU mL −1 .

Stored urine, sludge and manure
Undiluted stored human urine was obtained from the Swiss Federal Institute of Aquatic Sciences (Eawag) in Dübendorf, Switzerland (CH), where urine is collected from waterless urinals and men's NoMix toilets and women's NoMix toilets respectively. Three batches of male stored urine taken in 3 different years (2012, 2013, 2014) and one batch of female stored urine (2014) were obtained and used for experimentation. An additional batch of stored urine was collected from urine diverting dry toilets in Durban, South Africa (SA) in 2014. Diluted stored urine was obtained by mixing undiluted stored urine (male, CH, 2012) and MilliQ water at urine : water ratios of 1 : 1, 1 : 2 and 1 : 9. In total, the various stored urine batches and dilutions yielded 15 urine samples (U1-U15; Table 1). For experiments with sludge, three batches of sludge were used: two batches (S1, S3) consisted of a synthetic fecal sludge. This sludge was made of walnuts, straw flour, kaolinite, sodium phosphate, ammonium chloride and potassium nitrate, and was digested by an inoculum of bacteria obtained from a thermophilic anaerobic digester, as described by Gallandat et al. 20 An additional batch (S2) consisted of fecal sludge collected from septic tanks in Switzerland. Two batches of pig (M1, M2) and cow manure (M3, M4) were collected in the Swiss country-side.
Upon arrival in the lab, the stored urine, sludge and manure were stored at 4°C from weeks to months until use. Prior to characterization, all urine samples were centrifuged at 10 000 × g for 10 minutes. For sludge and manure characterization, 5-10 ml of MilliQ water were added, and the sample was shaken for 10-15 minutes and centrifuged at 4000 × g for 15 minutes. Stored urine, sludge and manure physical and chemical characteristics (summarized in Tables 1 and S1 †) were determined as follows: pH was measured at experimental temperature (780 pH Meter with primatrode with NTC no. 6.0228.010, Metrohm, Herisau, Switzerland); the total ammonium nitrogen (TAN; NH 4 + /NH 3 ) concentration was determined by ion chromatography (ICS-3000, IonPacCS16 column) with electrical conductivity detection (Dionex, Switzerland); phosphate, sulfate and chlorine concentrations were measured by ion chromatography (ICS-3000, IonPac AS11-HC column); magnesium, calcium, potassium and sodium by inductively coupled plasma optical emission spectrometry (ICP-OES, Ciros, Spectro Analytical Instruments, Kleve, Germany); soluble chemical oxy-gen demand (SCOD) with cuvette tests (Hach-Lange, Berlin, Germany) and total inorganic carbon (TIC) by means of a TOC-TN Analyser (IL 550, Hach-Lange, Berlin, Germany). For TAN measurements, samples were diluted in 0.01 M HCl and for TIC measurements in 0.01 M NaOH to avoid loss of NH 3 and CO 2 , respectively. For all other ions, samples were diluted in MilliQ water. Additionally, total solids (TS) were determined in sludge and manure according to standard methods. 21 Given the low TS (<7%), one Liter of sludge or manure was considered as one kg. This characterization was generally performed in quadruplicate for stored urine and in triplicate for sludge and manure (see below for details). For male stored urine (2012) (U1-U11, see Table 1), the ion content was determined in the undiluted sample (U1) only, and was extrapolated for the rest of the samples composed of this urine (U2-U11). Only pH and TAN were determined under each experimental condition (U1-U11; see Table S1 †).
Ion activities in each matrix were determined as a function of experimental temperature, pH and solution composition using PHREEQC (version 2.18.00) 22 and a database using the Pitzer approach for calculating ion activities. 23 The concentrations expressed in mol kg −1 in PHREEQC were considered equivalent to mol L −1 . SCOD was transformed to acetate equivalents, because acetate was shown to represent 47% of the SCOD in stored urine. 24 The transformation was determined according to the following stoichiometric relation:

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Manure was additionally tested for the presence of somatic and F-RNA coliphages. Only somatic coliphages were detected in pig manure at >10 3 PFU g −1 .

Experimental setup
ΦX174 and T4 were tested in one batch of stored urine, one batch of sludge and one batch of manure; HAdV in two batches of stored urine, one batch in sludge and one batch of manure; and MS2 in all the batches described in Table 1. An overview over the experimental setup is provided in the supplementary material (Scheme S1 †). Prior to each inactivation experiment, the matrices were stored at the targeted experimental temperature during one (urine) or five to ten (manure, sludge) days to establish a stable temperature and microbial activity as well as to avoid drastic changes in solution composition during the inactivation phase.
Stored urine experiments. For experiments in stored urine, one milliliter of a MS2, ΦX174, T4 or HAdV solution containing 10 7 -10 10 PFU or MPNCU mL −1 in virus dilution buffer (VDB; 5 mmol L −1 NaH 2 PO 4 , 10 mmol L −1 NaCl, pH 7.5) was added to airtight 116 ml glass serum flasks (Infochroma) containing 114 ml of stored urine solution. One mL samples were periodically withdrawn from each flask with a sterile syringe and were filtered through a 0.22 μm filter (Millipore).
Sludge and manure experiments. For sludge and manure, 1 mL of a MS2, ΦX174, T4 or HAdV solution (10 7 -10 10 PFU or MPNCU mL −1 in VDB) was added to 150-300 ml of sludge or manure. The suspension was stirred during 5 minutes in a 500 mL closed bottle and 12 mL aliquots were then transferred into 15 ml falcon tubes (Sarstedt, Nümbrecht), which were tightly closed with a cap and parafilm. Two grams of samples were periodically collected from sacrificial falcon tubes, and were weighed and mixed with 10 ml beef extract solution (BES; 100 g L −1 beef extract (Merck), pH 7.2) to promote the elution of viruses from solids. Samples were then shaken for 10-15 minutes and centrifuged for 20 minutes at 4000 × g at room temperature. 25 Preliminary experiments yielded a 95% recovery of MS2 spiked in sludge with this procedure. 1 ml of the supernatant was then withdrawn with a sterile syringe and filtered through a 0.22 μm filter (Millipore).
Buffer experiment. HAdV inactivation was further assessed in phosphate carbonate buffer (50 mmol L −1 carbonate and 40 mmol L −1 phosphate) at pH 9.0 and 35°C as described previously. 15 Enumeration. The filtered urine, sludge or manure samples were directly diluted in medium containing 2% FBS (HAdV) or VDB (MS2, ΦX174, T4), and were stored at 4°C for no more than six hours prior to enumeration. Each stored urine was tested in duplicate flasks and sludge and manure were tested in triplicate tubes for all organisms. Phage titers were determined in duplicate or triplicate from the same reactor, whereas HAdV was enumerated once per reactor.
Testing the role of microbiological activity and cations. The effect of HEAM-associated parameters on inactivation was tested in stored urine and manure. Urine-specific components were investigated in samples of 1 : 9 diluted stored urine (U10) with MS2. To test the role of microbial activity, MS2 was exposed to samples filtered at 0.22 μm to remove live microorganisms. To inhibit enzyme activity, samples were first filtered at 0.22 μm to remove microorganisms and the remaining enzymes were heat-inactivated at 65°C for 30 minutes. To minimize the influence of cations on inactivation, the complexing agent ethylenediaminetetraacetic acid (EDTA; Acros) was added to stored urine to obtain a final concentration of 10 and 50 mmol L −1 . The role of microbial and enzyme activity in manure (M4) was tested by exposing MS2 to heat-sterilized manure (65°C for 1 h).

Data analysis
Inactivation kinetics were determined by least-square fit of the data to a first-order model: where C 0 and C [PFU or MPNCU mL −1 ] are the virus concentrations at time 0 (initial) and t, and k obs is the first-order inactivation rate constant [day −1 ]. Non-first-order inactivation kinetics were fitted to a biphasic model: where C 0,fast and C 0,slow indicate the initial concentrations, and k obs,fast and k obs,slow the inactivation rate constants for the fast-and slow-inactivating populations, respectively.

Model to predict MS2 inactivation
MS2 inactivation rate constants were determined in all stored urine, sludge, and manure samples, over a wide range of solution conditions and experimental temperatures. This data set was used to challenge the predictive model of MS2 inactivation discussed in Decrey et al., 15   where the parameter β is the Brønsted coefficient, and D is a constant, both specific to MS2. For MS2 inactivation, we observed β = 0.41 at 35°C. To predict inactivation at different temperatures, the temperature-dependence of each parameter in the Brønsted catalysis law (eqn (4)) must be considered. However, literature reports on a similar reaction, namely the base-catalyzed decomposition of nitramide, showed no relevant temperature dependence of the Brønsted plot slopes for a temperature range from 15-45°C. 26,27 Correspondingly, in our studies, the coefficient β determined experimentally at 20°C and 35°C were equal (see ESI, † Fig. S1). We therefore assumed a constant value of β over the temperature range considered. In contrast to β, pK a , k j and D are dependent on temperature. For any given temperature, DĲT) was estimated based on available experimental data 15 for j = NH 3 as follows: All pK a ĲT) values were calculated according to eqn (S1) (see ESI, † determination of pK a ), and k NH 3 ĲT) was determined by the Arrhenius relationship reported in Decrey et al.: 15 (6) Finally, it is apparent that only those bases contribute to inactivation of MS2 that are present at a significant activity (eqn (3)), and that have a conjugated acid with a relatively high pK a (eqn (4)). Given the composition of the matrices used herein (Tables 1 and S1 †), the only bases (j) considered were therefore OH − , NH 3  . Note that a user-friendly interface for the predictive model is available for free online (https://lodecrey.shinyapps.io/ MS2inactivation/). 28 3 Results and discussion

Inactivation kinetics
Example virus inactivation curves obtained in one batch of human urine, sludge and manure are shown in Fig. 1 (see ESI, † Fig. S2 and S3 for the complete set of inactivation curves). Viruses in all matrices studied exhibited a loss of infectivity with time, and inactivation followed first-order kinetics, except in the case of ΦX174 and T4 in manure ( Fig. 1 and  S3 †). First-order inactivation rate constants and coefficients of determination are listed in Table S2 (ESI †). First-order inactivation kinetics were also reported by Höglund et al. 29 and Vinnerås et al. 30 for Salmonella phage 28B and rotavirus in stored urine. In contrast to the findings herein, however, Vinnerås et al. 30 observed a two phase inactivation behavior for MS2 and ΦX174, with an initial fast inactivation followed by a slow first-order reduction.
The first-order inactivation kinetics observed herein indicate that solutions conditions were stable over the course of the inactivation experiments. Correspondingly, a characterization of the composition of stored urine over time showed that both pH and ion concentrations were stable when stored at 20°C or 35°C. For sludge and manure, however, changes in solution conditions were observed (ESI, † Table S3). For example, in the case of sludge (S2), the concentration of TAN doubled over the course of 14 days, and the concentrations of Ca 2+ , Mg 2+ , SO 4 2− decreased more than two-fold whereas the pH and other ions were stable; in the case of manure (M1), the pH increased from 7.58 to 8.0 and TIC doubled whereas other ions were stable. The effect of the changing solution conditions on inactivation, however, remained small: predicted inactivation rate constants of MS2 (see section 2.6) differed by less than a factor of 1.4 for most matrices over the duration of an experiment (see ESI, † Table S3). Consequently, for any downstream analysis, the average of the initial and final composition was used for S2, S3, M1, M2 and M3 (Tables 1 and S1 †); for S1 and M4, only the composition at initial time was assessed.

Validation of MS2 inactivation prediction in stored urine, sludge and manure
The comparison between predicted and observed inactivation rate constants is shown in Fig. 2. Over half of the predictions

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fell within 80-120% of k obs , and three-fourths of the predictions were within 60-140% of k obs . This ability of the model to accurately predict MS2 inactivation supports the notion that the dominant underlying mechanism of inactivation is basecatalyzed transesterification of the ssRNA genome. 15,16 This appears to be the case in both synthetic solutions and in real matrices such as stored urine, and sludge and manure. Consistent with our results, Gao et al. 31 recently reported that during mesophilic anaerobic treatment of Coxsackievirus, the viral genome was the main inactivating target. A model sensitivity analysis was conducted to assess the influence of pH and temperature, and the inclusion of measured ion concentrations in the model, on the accuracy of the prediction (Fig. S4 †). Specifically, we re-assessed the model prediction for all 22 samples at either pH values of 0.1 units surrounding the measured value or at temperatures of 1°C surrounding the measured temperature. In addition, predictions were carried out that included only TAN, or TIC and TAN, but none of the other ions in solution. This analysis revealed similar sensitivity to shifts resulting from changes in pH by 0.1 units and from changes in temperature by 1°C (Fig. S4 and Table S2 †). A relatively minor error in the measurement may thus lead to an inaccurate k pred . Interestingly, no relevant differences were observed if all ions were taken into account in the prediction, as compared to only the TIC and TAN (Fig. S4 and Table S2 †). Thus, measurements of temperature, pH, TIC and TAN are sufficient to obtain a reasonably accurate prediction of MS2 inactivation rate constants. Removal of TIC from the prediction led to a lower accuracy ( Fig. S4 and Table S2 †). This highlights the importance of carbonate in MS2 inactivation in HEAM. Carbonate and bicarbonate contributed between 15 and 40% to the total k obs in stored urine, sludge and manure (Fig. 3). The contribution of carbonate species was even higher (>50%) in sludge S2, which had a pH < 8.0 and equivalent amounts of TIC and TAN.

Influence of HEAM-associated parameters on MS2 inactivation
Despite the quite high accuracy of the predicted inactivation rate constants for MS2 in all matrices considered, some small deviations from the measured k obs were observed: for some stored urine samples, the model yielded a slight overestimate of the rate constant compared to the observation, while the model tended to underestimate inactivation in 1 : 9 diluted urine, sludge and manure (Fig. 2). These deviations may be in part linked to imprecise measurements of the ion concentrations and the PHREEQC estimation of the ion activities. Alternatively, underestimations could indicate that additional inactivating processes occur that are not accounted for in the model. In this context, we assessed the role of two HEAM-associated parameters in inactivation, namely biological (microbial or enzymatic) activity and metal ions, and we discussed the influence of additional bases not considered by our model.
3.3.1 Microbiological activity. Stored urine is known to possess bactericidal properties, 12,13,32 which may limit the effects of microbiological activity on viruses in undiluted urine. Upon urine dilution, however, the concentration of bactericidal substances diminishes, such that a more active microbial population may be established. Consequently, the effect of microbiological activity on virus survival could be greater in diluted than in undiluted urine. Such a scenario would account for the under-predicted inactivation rate constant in diluted urine (Fig. 2). However, neither filtration to remove microorganisms, nor heating of the diluted urine to inactivate enzymes, affected the observed MS2 inactivation kinetics  (3) and (4). The solid line represents a 1 : 1 relation between measurement and prediction (k pred /k obs = 1). Dashed lines indicate 80% and 120% of k pred /k obs (i.e., k pred /k obs = 0.8 and 1.2 respectively). The inset shows k pred versus k obs for different dilutions of urine. The urine : water ratio for each data point is indicated. ( Fig. 4, green bars). Hence, microbiological activity could not account for the differences between k obs and k pred in 1 : 9 diluted urine. Heat-sterilization of manure (M4) even led to an increase in inactivation, resulting in a k obs of 4.6 (±0.7) day −1 (data not shown) compared to 2.7 (±0.8) day −1 in untreated manure. Thus, MS2 seemed insensitive to biological activity. Similar results were observed in soil saturated with secondary effluent by Nasser et al., 33 and in nitrifying urine by Bischel et al. 34 In contrast, Mondal et al. 35 reported that MS2 could be inactivated by both commercial and sludge-derived protease solutions. At sufficiently high protease concentrations, biological activity may thus compete with chemicallymediated MS2 inactivation in HEAM, though this situation was not encountered herein.

Metal ions.
Metal ions are known to accelerate base-catalyzed RNA transesterification, 36,37 and may thus enhance MS2 inactivation in HEAM compared to laboratory solutions. This transesterification-accelerating mechanism is currently not considered in k pred . To reduce the effect of free metal ions, 1 : 9 diluted urine was amended with EDTA, a metal complexing agent. This was found to decrease k obs to a value close to k pred (see Fig. 4, orange bars). Thus, the presence of EDTA suppressed the action of the urine constituents responsible for the higher k obs . Interestingly, if ammonia (as NH 4 Cl) was added to (EDTA-free) 1 : 9 diluted urine, k obs increased as expected based on the increase in {NH 3 } (see ESI, † Fig. S5). The effect of ammonia was thus additive to that of the metal ions, resulting in an overall higher inactivation than in other urine matrices with the same NH 3 activity. We currently cannot explain, however, why urine dilution, followed by ammonia addition results in higher inactivation than an equivalent ammonia activity in undiluted urine.
The role of metal ions was not explicitly studied in sludge and manure. Nevertheless, literature reports indicate that particularly manure contains metal cations up to the mmol L −1 concentration range. 38,39 It is thus reasonable to conclude that metal ions in these matrices also contribute to MS2 inactivation. ). Odorous compounds such as sulfide (pK a1,2 = 6.99, 12.92) and 4-methylphenol (pK a = 10.6), which have high pK a values may also contribute to the base-catalyzed inactivation. The concentration of total sulfide and 4-methylphenol were shown to be in the mmol L −1 range in stored urine and fecal sludge. 40,41 The effect of these bases on inactivation remains to be tested.

Influence of genome type and microbial activity on virus inactivation kinetics in urine, sludge and manure
As discussed above, considerable heterogeneity exists among the inactivation kinetics of viruses with different genome types. Specifically, in well-controlled solutions, DNA viruses were shown to be more persistent than ssRNA viruses. To validate this trend in real matrices, we compared the inactivation of ssRNA MS2 phage to that of three DNA viruses, specifically phage ΦX174 (ssDNA), phage T4 (dsDNA) and HAdV (dsDNA). Inactivation kinetics were assessed in a subset of stored urine, sludge and manure samples at 35°C, and were compared to those obtained in laboratory solution with and without NH 3 . 15,16 As expected, the ssRNA virus MS2 was inactivated more readily than the DNA viruses in most matrices, (Fig. 1 and ESI † Table S2). This confirmed the higher sensitivity of ssRNA viruses to NH 3 and mildly alkaline pH. In undiluted stored urine at 35°C (U12), a four log 10 (99.99%) inactivation was achieved within one day for MS2, whereas it took more than 100 days to reach the same level of inactivation for T4. In sludge and manure the differences among ssRNA and DNA viruses were smaller, with a four log 10 loss being achieved within 15 and 207 days in sludge (S2) and 3.5 and 40 days in manure (M4) at 35°C for MS2 and T4 respectively. A possible reason for the narrower range of inactivation kinetics among ssRNA and DNA viruses in sludge and manure is the contribution of microbial or enzymatic activity to inactivation. While microbiological inactivation is not relevant for MS2 compared to its rapid chemical inactivation (see section 3.3.1), it may accelerate the inactivation of DNA viruses beyond the slow chemical inactivation kinetics in HEAM, in particular in matrices with high microbiological activity.
Consistent with our previous findings, HAdV was the most readily inactivated among the DNA viruses, and it was more resistant than MS2 in stored urine (U4) at pH 8.2 and approximately 20 mmol L −1 NH 3 and in manure (M4) (Fig. 1 and ESI † Table S2). However, in stored urine with a higher pH and NH 3 content (U12) and in sludge (S2), the inactivation of Fig. 4 MS2 inactivation rate constants determined at 35°C in 1: 9 diluted, raw or modified urine. Modifications were filtration and heating to remove microbial or enzymatic activity (green), and addition of EDTA to complex metal ions (orange). Also shown are the rate constants in raw urine k obs (yellow) and the predicted rate constant k pred (grey). k norm corresponds to the ratio of the k obs determined in the modified urine samples and k obs in raw 1 : 9 diluted urine (yellow). Error bars depict 95% confidence interval associated with k norm .

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HAdV increased, resulting in similar to greater inactivation rate constants compared to MS2 ( Fig. 1 and ESI † Table S2). A comparison with controlled solutions of similar physicalchemical properties revealed that pH could account for the fast inactivation observed in U12 (Fig. 5). In contrast, inactivation in S2 could not be explained by the known physicalchemical parameters, indicating a potential contribution of microbiological processes. Faster than expected inactivation was also observed for ΦX174 and T4 in manure. Specifically, these two phages exhibited biphasic inactivation behavior in manure with an initial fast inactivation followed by a secondary, slower phase (eqn (2); ESI, † Fig. S6). For both phages, k obs_fast was 4-5 times larger than k obs_slow (Fig. 5, green bars) and the k obs_slow generally corresponded to the k obs in a controlled solution with the same physical-chemical properties (Fig. 5, grey bars). The first, rapid decrease in infective virus thus is caused by parameters associated with manure that are not present in laboratory solutions, whereas the second, slower phase may be attributed to the effect of solution conditions only. We propose that the biphasic inactivation kinetics is associated with virus adsorption onto the manure. Specifically, while irreversible adsorption can be ruled out as a removal mechanism (given the high recovery of infective virus in our experimental protocol; see Materials and methods), adsorption to solids may lead to the protection of viruses from microbial and enzymatic activity. The fast, initial inactivation could thus be dominated by microbiological inactivation of suspended viruses, whereas the slower phase results from physical-chemical inactivation of viruses adsorbed to solids. This hypothesis is consistent with the findings by others that demonstrate faster inactivation in the liquid-than in the solid-associated virus fraction during anaerobic digestion 42 and in wastewater. 43 Microbiological contributions to virus inactivation have previously been reported for HAdV, Hepatitis A virus, norovirus and enteroviruses in a range of matrix types. 33,[44][45][46][47][48][49][50][51] In most cases, inactivation could only be partly attributed to biological processes, whereas physical-chemical matrix components also played a role. Unfortunately, the matrix composition was usually poorly characterized, and it is therefore difficult to parameterize its influence in inactivation. It appears, however, that the most important matrix components and their resulting virucidal activities vary widely depending on both the matrix and virus type. For example, it was shown that septic tank effluent digestion was more efficient at inactivating viruses when mixed with dairy cattle or swine manure slurry. 50,51 The same authors observed that among 31 bacterial strains isolated from animal manure, only 10 proved to be efficient at inactivating virus. Here, we propose inactivating microbial or enzymatic activity in sludge but not in manure for HAdV, in manure but not in sludge for T4, and in both for ΦX174 (Fig. 5). The distinct matrix and virus parameters that result in inactivation, however, remain to be determined.
The complex nature of virus inactivation in real matrices is further reflected in the literature, where a large variation in virus inactivation kinetics in excreta is reported. Consequently, results contradicting our data can be found: for example, others have shown ΦX174 to be inactivated as fast as MS2 in stored urine 12 and stored fecal sludge, 14 and HAdV to be inactivated faster than MS2 in fecal sludge. 52 Similarly, ssRNA phage f2 and coxsackievirus exhibited comparable inactivation to rotavirus (dsRNA) during mesophilic anaerobic digestion of sludge with, albeit at low pH (~7.3). 53 On the other hand, other studies provided observations consistent with our data. For example, F-RNA specific coliphages were shown to be more sensitive than somatic (DNA) coliphage and dsDNA Salmonella phage 28B during mesophilic digestion of raw sewage sludge 54 and the organic fraction of municipal solid waste. 55 Furthermore, somatic coliphages were found to exhibit low sensitivity to the addition of urea, calcium carbonate and sodium percarbonate used to sanitize composted sewage sludge, which is consistent with our finding that ΦX174 and T4 are not affected by the main chemical components of stored urine and sludge. 56 Overall, the influence of solids content on the inactivation of different virus types remains poorly understood and needs to be further elucidated. In particular, more research is needed on matrices  Table S2. † For ΦX174 and T4 in M4, columns with diagonal patterns depict k obs_slow . Error bars depict 95% confidence interval associated with k obs . with lower liquid fractions, such as fecal sludge (solids content between 20-95% (ref. 14)).

Conclusions
Among enteric viruses, the vast majority have an ssRNA genome. A good understanding of the factors that promote the inactivation of ssRNA viruses during waste treatment is therefore particularly important. The data presented herein demonstrate that for ssRNA phage MS2, inactivation kinetics and mechanisms established in laboratory solutions are transferable to real matrices. We furthermore established that carbonate, ammonia, pH and temperature are important drivers of inactivation that need to be maximized to enhance treatment efficiency. Future studies should be extended to additional ssRNA viruses to more conclusively establish the optimal treatment conditions. While viruses with other genome types are not as common, some enteric viruses with public health relevance have DNA and dsRNA genomes (e.g., adenovirus, polyomavirus, rotavirus). For DNA and dsRNA viruses, kinetic insights developed in controlled solutions may not be as readily transferable to real matrices, due to the potential contribution of microbiologically driven inactivation processes in HEAM. The kinetics based on physical-chemical parameters established in laboratory solutions can therefore only be considered as worst case scenarios for the inactivation of DNA and dsRNA viruses in HEAM.
Discrepancies exist between our results and literature reports, as well as among different literature reports. To reconcile those, a better understanding of the mechanisms involved in virus inactivation in HEAM is needed. While we believe that we have a good handle on main mechanism involved in the inactivation of ssRNA viruses, those responsible for DNA and dsRNA virus inactivation remain to be determined. In this context, we again emphasize the potential contribution of microorganisms on virus inactivation. Determining the virus properties that render it susceptible to microbiological inactivation will be an important next step in understanding inactivation in HEAM.
Finally, this study helps identify appropriate proxies to monitor virus inactivation in HEAM. Specifically, our data confirms that somatic coliphage such T4 or ΦX174 are conservative indicators of resistant ssDNA, dsDNA and dsRNA viruses. However, they are too stable to serve as indicators for the inactivation of the more labile HAdV. The utility of these indicators as suitable proxies for further DNA or dsRNA viruses should thus be confirmed.


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Tanshinone IIA improves functional recovery in spinal cord injury-induced lower urinary tract dysfunction

Tanshinone IIA, extracted from Salvia miltiorrhiza Bunge, exerts neuroprotective effects through its anti-inflammatory, anti-oxidative and anti-apoptotic properties. This study intravenously injected tanshinone IIA 20 mg/kg into rat models of spinal cord injury for 7 consecutive days. Results showed that tanshinone IIA could reduce the inflammation, edema as well as compensatory thickening of the bladder tissue, improve urodynamic parameters, attenuate secondary injury, and promote spinal cord regeneration. The number of hypertrophic and apoptotic dorsal root ganglion (L6–S1) cells was less after treatment with tanshinone IIA. The effects of tanshinone IIA were similar to intravenous injection of 30 mg/kg methylprednisolone. These findings suggested that tanshinone IIA improved functional recovery after spinal cord injury-induced lower urinary tract dysfunction by remodeling the spinal pathway involved in lower urinary tract control.

Introduction
Lower urinary tract (LUT) dysfunction after spinal cord injury (SCI) has been recognized as one of the severe complications for SCI patients, even of higher importance than the loss of locomotion (Pikov and Wrathall, 2001;Leung et al., 2007). Urine storage and micturition are complex in the normal condition, requiring the spinal cord to integrate information from the brain, bladder, and urethra (Yu et al., 2003;Birder et al., 2010). SCI impairs LUT by interrupting the communication between the cerebral and spinal circuits that coordinate the bladder detrusor and external urethral sphincter, leading to a severe disorder known as detrusor-sphincter dyssynergia (de Groat and Yoshimura, 2012;Gao et al., 2015). Therefore, the bladder cannot empty efficiently, which inevitably causes more serious consequences, such as urinary tract infections, urinary calculus, hydrone-

Tanshinone IIA improves functional recovery in spinal cord injury-induced lower urinary tract dysfunction
DRGs (L6-S1) at 4 weeks after SCI Histology of bladder tissue Spinal cord regeneration Dorsal root ganglion (L6-S1) cells Urodynamics Spinal cord at 4 weeks after SCI Urodynamic assessment Bladder weight LUT dysfunction after SCI SCI rats were treated with an intravenous injection of TIIA/MP phrosis, chronic renal failure and uremia.
Most of the research in LUT dysfunction recovery after SCI focuses on adjusting the bladder or sacral nerves with drug intervention, surgical therapies and functional electrical stimulation. However, these therapeutic methods are less than satisfactory. First, the standard treatment for neurogenic bladder after SCI is usually limited to clean intermittent catheterization, which can lead to repeated urinary tract infection and lower quality of life (Jamison et al., 2013). Second, the antimuscarinic drugs that are currently the first-line choice for the treatment of neurogenic detrusor overactivity cannot be used chronically, because the high dosage required in patients with neurogenic detrusor overactivity often results in more severe side effects, such as dry mouth, constipation, blurred vision, drowsiness, and dry skin and mucosa (Cameron, 2010;Goldmark et al., 2014). Furthermore, surgical options for the neurogenic bladder, such as augmentation cystoplasty, may solve the problems related to bladder capacity but would lead to urinary tract infection, mucus production, urolithiasis and other complications (Kikuno et al., 2009;Lee et al., 2013). Functional electrical stimulation offers another approach to restore LUT function by activating the bladder detrusor and inhibiting the urethral sphincter to produce voiding, alternatively, inhibiting the bladder detrusor to provide urinary continence. Dorsal rhizotomy, before functional electrical stimulation, that transects the dorsal spinal roots to eliminate unwanted bladder and urethral reflexes also eliminates desirable reflexes that affect sexual and bowel functions (Ho et al., 2014).
Tanshinone IIA (TIIA) is an important lipophilic diterpene extracted from Salvia miltiorrhiza Bunge and has been widely used in traditional Chinese medicine for the treatment of many diseases, especially in cardiovascular and cerebrovascular diseases (Xu and Liu, 2013). TIIA also has neuroprotective effects through its anti-inflammatory, anti-oxidative and anti-apoptotic properties Gao et al., 2012;Su et al., 2012;Yan et al., 2012). Therefore, we hypothesized that TIIA has great potential in remodeling the spinal pathway. The primary purpose of this study is to evaluate the effect of TIIA in reorganizing the spinal pathway related to LUT control.

Animals
A total of 80 specific-pathogen-free female Sprague-Dawley rats, aged 8-10 weeks and weighing 220-250 g, were obtained from Vital River Laboratories in Beijing, China (animal license No. SCXK (Jing) 2012-0001). The rats were housed 3 to 4 per cage, kept on a 12-hour light/dark cycle, and allowed free access to food and water. The experimental protocol was approved by the Animal Care and Use Committee of Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, China (approval No. 2014-14). Adult Sprague-Dawley rats (n = 80) were equally and randomly divided into four groups: sham, SCI, TIIA (SCI + TIIA) and methylprednisolone (MP) (SCI + MP).

Establishment of SCI models
Rats were intraperitoneally anesthetized with 10% chloral hydrate (3.5 mL/kg) and a midline dorsal incision was made over the lower thoracic vertebra to expose the vertebral spines and paravertebral muscles. A laminectomy was made at T 9-11 to expose the dura. A standardized mild, incomplete contusive SCI was produced using the multicenter animal spinal cord injury study device (made by New York University, New York, USA) with a 10-g weight dropped from a height of 25 mm onto the exposed dura (Gruner, 1992). The muscle and skin overlying the wound were then sutured in layers, followed by a 3 mL subcutaneous injection of saline solution to replace the blood volume lost during the surgery. After surgery, rats were placed on a heating pad until they awoke. The Basso, Beattie & Bresnahan open field locomotor test and lower abdominal palpation were used to test the success of the SCI model. When the Basso, Beattie & Bresnahan score was 0 (no observable hindlimb movement) and the bladder was excessively filled the rat model was considered successful. Subsequently, the rats were injected with penicillin subcutaneously (200,000 unit/animal/day) for 3 days to prevent incision and urinary tract infections. Bladders of the injured rats were gently manually expressed 2 to 3 times daily by Crede's method, with the volume of expressed urine recorded, until spontaneous micturition was re-established. This provided an estimation of the time required for the re-emergence of spontaneous micturition (urine volume < 2 mL) (Leung et al., 2007). Infection, decubitus, dehydration and autophagia were monitored daily during the process.
Rats were given easy access to food and water and were placed in cages with highly absorbent bedding. Fluid intake was not controlled as it has been shown that this did not affect the time for the recovery of a reflex bladder (Chancellor et al., 1994). Rats failing to establish SCI models or those died were excluded from the experiment and replaced by the same number of rats.
The sham group consisted of rats that were only subjected to laminectomy.

Drug treatment
TIIA injection (batch No. A140133; No. 1 Biochemical Pharmaceutical Co., Ltd., Shanghai, China; 10 mg/2 mL) and MP injection (batch No. A08894; Pfizer Manufacturing Belgium NV, Belgium, 40 mg) were purchased from Dongzhimen Hospital, Beijing University of Chinese Medicine, China, the main component was sodium TIIA sulfonate or methylprednisolone sodium succinate, respectively. The rats in TIIA group were intravenously administered TIIA (20 mg/kg) once a day at the same time from day 1 to day 7 post-surgery. The dose and timing of TIIA administration used were the same as in previous studies (Yin et al., 2012). The rats in the MP group were intravenously administered MP (30 mg/kg) once, immediately post-surgery after confirming the success of the SCI model.

Urodynamic assessment
The urodynamic studies were carried out in awake rats at 2 and 4 weeks after SCI so that the bladder was stabilized after the spinal shock phase (D' Amico and Collins, 2012). To insert a catheter into the bladder, rats were briefly anesthetized with isoflurane. A PE-50 polyethylene catheter was inserted via a midline abdominal incision into the bladder through the bladder dome and secured with cotton thread. The intravesical catheter was connected via a three-way stopcock to a pressure transducer (MP150, BIOPAC Systems, Inc., Goleta, CA, USA) and a micro-infusion pump (WZ-50C6, Smiths Medical Instrument, Co., Zhejiang, China) to record intravesical pressure and infuse saline into the bladder, respectively. Intravesical pressure was recorded continuously using data acquisition software (BIOPAC AcqKnowledge4.2, BIOPAC Systems, Inc.). Anesthesia was stopped after catheter implantation and the rats awakened then left untouched for 30 minutes for bladder stabilization. After this period, rats received a continuous infusion of saline (0.9% NaCl; 37°C) at a rate of 0.1 mL/min (Smith et al., 2008;Artim et al., 2011). It was not necessary to measure abdominal pressure because the abdominal incision was not sutured.
The parameters we assessed included the micturition pres-sure (maximum bladder pressure during micturition), basal pressure (the lowest bladder pressure between micturitions), threshold pressure (bladder pressure immediately before micturition), and the intercontraction interval. The number of non-voiding contractions was also measured (Andrade et al., 2011). Saline voided from the urethral meatus was collected and the voided volume was measured. Saline infusion was stopped at the beginning of the voiding contraction to avoid interfering with the measurement of the residual volume. The residual volume was measured by drawing saline through the intravesical catheter and then manually expressing the remaining intravesical contents by exerting pressure on the bladder abdominal wall. The bladder capacity was calculated as the voided volume plus the residual volume. The voiding efficiency (%) is equal to (voided volume/bladder capacity) × 100% (Urakami et al., 2007;Andrade et al., 2011).

Sample drawing
At the end of the urodynamic experiment, the rats were intracardially perfused with saline followed by ice-cold 4% paraformaldehyde. Subsequently, bladders were removed, blotted dry, and weighed. Four dorsal root ganglions (DRGs) on both sides of L 6 -S 1 level, and the 5-mm spinal cord tissues including the central site of injured tissue (T 9 ) were collected. After fixing in 4% paraformaldehyde overnight, tissue blocks were embedded in paraffin. The bladders were cut into 5-µm slices and stained with Masson's trichrome. The spinal cords (T 9 ) were cut transversely into 9-µm slices and stained with Cresyl Violet for Nissl staining. The DRGs were cut transversely into 5-µm slices for terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay and 9-µm slices for Nissl staining.

Histological analysis
Hematoxylin-eosin staining Sections (5-µm thick) of DRGs at 4 weeks after surgery were deparaffinized and washed twice with distilled water and then immersed in hematoxylin solution for 3 minutes, fol-Normal bladder in sham group shows stable intravesical pressure without non-voiding contractions. SCI-induced hyperreflexic-bladder shows many large non-voiding contractions (more than 11 mmHg) during saline infusion. At 4 weeks, the micturition interval was more regular than at 2 weeks. After TIIA administration, bladder shows stable intravesical pressure without non-voiding contractions at 2 and 4 weeks after SCI.
After MP administration, bladder shows some non-voiding contractions during saline infusion at 2 weeks after SCI, but clearly improved at 4 weeks. SCI: Spinal cord injury; TIIA: tanshinone IIA; MP: methylprednisolone. Figure 2 Typical cystometrogram patterns of bladder activity during urodynamics at 2 and 4 weeks after SCI.

Figure 1 Bladder weights in four groups of rats at 2 and 4 weeks after SCI.
The bladder weights of SCI group, TIIA group and MP group increased significantly compared with sham group at 2 and 4 weeks after SCI. After drug administration of TIIA or MP, the bladder weights of the TIIA and MP groups increased less than those in the SCI group without any treatment (n = 5 in each group, *P < 0.05).  After washing with tap water for 30 minutes, the sections were dehydrated through 70%, 80%, 90%, 95%, and 100% alcohol, each for 2 minutes, and stained with eosin for 10 seconds. Finally, the sections were dehydrated through increasing concentrations of ethanol, cleared in xylene for 10 minutes and mounted.
Nissl staining Sections (9-µm thick) of spinal cord and DRGs at 4 weeks after surgery were deparaffinized and washed twice with distilled water. The sections were immersed in 5% Cresyl Violet for 5 minutes. The sections were then dehydrated in increasing concentrations of ethanol and mounted.
Masson's trichrome staining Sections (5-µm thick) of bladder tissue 4 weeks after surgery were deparaffinized and hydrated through graded alcohols to distilled water. The bladder tissues were stained with Harris Hematoxylin for 2-3 minutes, and then rinsed in distilled water until only the nuclei remained stained. The sections were then stained with Scarlet-Acid Fuchsin for 5 minutes, rinsed twice in distilled water, and immersed in phosphomolybdic acid for 5 minutes. Next, the sections were stained  TUNEL assay TUNEL assay was conducted in accordance with instructions of the TUNEL kit (Roche, Mannheim, Germany). 5-µm thick sections of DRGs at 2 weeks after surgery were washed with phosphate buffered saline (PBS, 0.01 M), incubated with proteinase K at 37°C for 30 minutes, washed with PBS, and then dried. Afterwards, samples were incubated with TUNEL reaction mixture at 37°C for 60 minutes, and washed with PBS. The slides of Nissl staining, Masson's trichrome staining and TUNEL assay were mounted and analyzed under a bright-field microscope (Nikon, Tokyo, Japan). The injured area in the transverse sections that were located in the lesion epicenter of spinal cord was measured by the Image-Pro plus 6.0 pathological analysis software (Media Cybernetics, Silver Spring, MD, USA). The mean diameter, cross-section areas, cell gaps, and the number of inflammatory cells in L 6 -S 1 DRG were counted and measured. The area of positive cell, mean density and integrated optical density in dorsal root ganglion sections were measured. The integrated optical density was equal to mean density × area of positive cells. The number of motor neurons in the anterior horn of spinal cord and the thickness of muscle layers and vascular congestion in the bladder tissue were all measured by this software. The ratio of injured area was equal to (the injured area/the cross-section of the spinal cord) × 100%.

Statistical analysis
Values were presented as the mean ± SEM. One-way analysis of variance was adopted for comparison between groups. Statistical analyses were performed using SPSS 20.0 statistical software (IBM, Armonk, NY, USA). A value of P < 0.05 was considered statistically significant.

Effect of TIIA on bladder weight after SCI
The weight of a rat's bladder indirectly reflects the extent of compensatory hypertrophy of the detrusor after SCI (Leung et al., 2007). The bladder weights of all the groups were measured at 2 and 4 weeks after surgery (Figure 1). The bladder weights of all experimental groups increased significantly compared with the sham group (P < 0.05), indicating the detrusor hypertrophied after SCI. After drug administration of TIIA or MP, the bladder weights of the TIIA and MP groups were remarkably lower in comparison with the SCI group (P < 0.05). However, there is no significant difference between TIIA and MP groups (P > 0.05).

Effect of TIIA on urodynamics after SCI
At 2 weeks after surgery, during the bladder-filling phase, the sham group showed a large number of voiding contractions in this phase. However, the SCI group showed more non-voiding contractions than the sham group (P < 0.05). The SCI group displayed marked alterations, such as reductions in intercontraction interval, voided volume and voiding efficiency. There were also increases in basal pressure, threshold pressure and bladder capacity compared with sham group (P < 0.05; Table 1). On the other hand, the Values are expressed as the mean ± SEM (n = 8 in each group). #P < 0.05, vs. sham group (one-way analysis of variance) and *P < 0.05, vs. SCI group (one-way analysis of variance). a The mean of 10 cycles was calculated for each rat, with the final result given as the average of all animals in an experimental group. b The number of non-voiding cycles within a 10-minute period. c Voiding efficiency was determined using the voided volume and residual volume from the last voiding cycle. SCI: Spinal cord injury; TIIA: tanshinone IIA; MP: methylprednisolone.
TIIA group exhibited significant improvements compared with the SCI group, such as higher intercontraction interval, voided volume and voiding efficiency (P < 0.05). There were also fewer non-voiding contractions, residual volume and bladder capacity (P < 0.05). The MP group also displayed higher voided volume and voiding efficiency (P < 0.05, vs. SCI group; Table 1). After 4 weeks, similar improvement can also be observed in the TIIA group. Some recovery of bladder function was more obvious after 4 weeks than 2 weeks compared with SCI only (Figure 2).

Effect of TIIA on secondary injury and spinal cord regeneration after SCI
The results of Nissl staining at 4 weeks after surgery are shown in Figure 3. First, compared with the SCI groups, the number of motor neurons in the anterior horn of the sham group was greater and their Nissl bodies and nuclei were clearly visible. The number of motor neurons in the anterior horn in SCI group had decreased dramatically 4 weeks after injury. However, the administration of TIIA or MP lessened this change over the same time. Second, with the development of secondary injury after SCI, the area of injured spinal cord gradually increased. In the TIIA and MP groups the ratio of injured area was significantly less than that of the SCI group (P < 0.05).

Effect of TIIA on histology of bladder tissue after SCI
The results of Masson's trichrome staining at 4 weeks after surgery were shown in Figure 4. Bladders from the SCI group revealed the presence of vascular alterations, edema, and proliferation of urothelial layers compared with bladders from the sham group. The continuity of the umbrella cell layer of the urothelium was disrupted in rats at 4 weeks after SCI. A marked neutrophil infiltration to the suburothelial tissue as well as blood vessel congestion and dilation were observed. However, there was less neutrophil infiltration to the suburothelial tissue in the TIIA or MP groups and the muscle layers were not as thick as in the SCI group (P < 0.05).

Effect of TIIA on the change of DRG (L 6 -S 1 ) cells after SCI
The isolated DRGs were examined histologically with Nissl staining and hematoxylin-eosin staining (Figure 5). First, almost no inflammatory cells were observed in the sham group, but there were a large number of inflammatory cells in the SCI group. In the TIIA or MP groups, only some of the ganglion cells were infiltrated by inflammatory cells. Second, the sham group showed typical morphology of normal ganglion cells; SCI group revealed that ganglion cell gaps widened significantly. With the administration of TIIA or MP, the ganglion cell gaps in the TIIA and MP groups were narrowed compared with the SCI group (P < 0.05). Third, the mean diameter and area of DRG cells in cross-section from SCI group was much higher than that of sham group and TIIA and MP groups (P < 0.05). Moreover, the normal cell nuclei in the sham group were round and centered; however, the cell bodies in the SCI group became hypertrophic and elongated with some of the nuclei shrunken or disap-peared. Some Nissl bodies also disappeared or were replaced by vacuoles. In the TIIA and MP groups, only a small number of cells were observed with shrunken or absent nuclei and absent or vacuous Nissl bodies. The integrated optical densities of apoptosis in L 6 -S 1 DRGs of TIIA and MP groups were significantly less compared with SCI group at 2 weeks after surgery (P < 0.05). There was no obvious difference in the integrated optical density of apoptosis between the TIIA and MP groups (P > 0.05; Figure 6).

Discussion
Urinary complications are very common in patients with SCI, therefore they attract considerable attention. Over the years, most of the therapeutic strategies have concentrated on the upper urinary tract and the improvement of the quality of life by facilitating the storage of urine and bladder emptying (Birder et al., 2010;Cruz and Cruz, 2011). However, the therapeutic effects are far from satisfactory. Recently, many studies have focused on remodeling the spinal pathway related to LUT control (Lee et al., 2013;Kim et al., 2015). SCI at the cervical or thoracic levels disrupts the voluntary control of micturition as well as the normal reflex pathways that coordinate detrusor and sphincter function. The bladder is initially areflexic, but then becomes hyperreflexic due to the emergence of a spinal micturition reflex pathway. Therefore, the bladder does not empty efficiently because of the loss of coordination between the bladder and urethral outlet, which is called detrusor-sphincter dyssynergia (Leung et al., 2007). Initially detrusor-sphincter dyssynergia after SCI causes bladder outlet obstruction and bladder load increase, leading to chronic inflammation and tissue edema. Then bladder weight increases and bladder wall gradually thickens. Other changes after SCI include the disruption of the continuity of the umbrella cell layer in urothelium and there is a marked neutrophil infiltration to the suburothelial tissue and blood vessel congestion and dilation. Ikeda and Kanai (2008) using optical imaging techniques showed that alterations at the luminal surface of the bladder contribute to the changes in LUT function after SCI. On the other hand, de Groat and Yoshimura (2012) found that detrusor-sphincter dyssynergia after SCI induce somal hypertrophy of bladder afferent neurons in the DRGs (L 6 -S 1 ). Their studies in cats proved that unmyelinated C-fiber afferents rather than Aδ afferents initiate voiding after SCI. Therefore, SCI not only causes changes in the structure of the bladder itself, but also leads to changes in the neuronal pathways innervating the LUT. These findings suggest that approaches that facilitate the remodeling of the spinal micturition reflex pathway might be useful in treating detrusor-sphincter dyssynergia after SCI.
It has been revealed that the recovery of bladder function after SCI could be mediated by improving the remodeling of the spinal pathway related to LUT control (Zhao et al., 2015). A popular area of research has been the use of cellular implants or the control of the extracellular environment of the injured site. It was found that injection of neural stem cells at the lesion site in the spinal cord of SCI rats enhanced threshold pressure, residual volume and non-voiding contractions (Kim et al., 2015). Olfactory ensheathing cells have also been used in some studies (Fouad et al., 2009). The technique of regulating the extracellular environment of the lesion site and the formation of the glial scar has also been useful in improving bladder function. For example, chondroitinase, which has been used alone or in combination with Schwann cell transplantation, improves recovery of bladder function in rats after SCI (Alluin et al., 2014). Other therapies have combined implants of biomaterials with other approaches mentioned above (Lee et al., 2013).
Our previous study demonstrated that TIIA can facilitate the recovery of neurological function in rats after SCI, and especially promotes the recovery of the sensory function of the hindlimb. Therefore, we hypothesized that the use of drugs for spinal cord repair may also improve the recovery of bladder function after SCI. In this study, detrusor-sphincter dyssynergia following SCI caused bladder outlet obstruction and bladder load increase, which result in gradual thickening of bladder wall and bladder weight increase. The difference of bladder weight between SCI group and sham group suggested that bladder wall thickening is a chronic compensatory action that increases with time (de Groat and Yoshimura, 2012). Tissue edema induced by inflammation contributes to the increase in bladder weight after SCI. After TIIA or MP was administrated to SCI rats, their bladder weight decreased compared with the SCI group. Nevertheless, there is no significant difference between TIIA and MP groups. The urothelial layers were also changed after SCI. The continuity of the umbrella cell layer of the urothelium is disrupted in rats at 4 weeks after SCI. A marked neutrophil infiltration to the suburothelial tissue and vascular congestion and dilation was observed. The changes can be attenuated by treatment with TIIA or MP.
Alterations at the luminal surface of the bladder may contribute to the changes in LUT function after SCI. Optical imaging techniques revealed that phasic contractions in bladders from SCI animals originate from localized sites in the bladder dome and are driven by activity arising in the urothelium (McCarthy et al., 2009). Removal of the mucosa eliminates the phasic contractions in bladders from SCI rats. In addition, gap junction blockers suppress the phasic contractions. It has been proposed that phasic activity in bladders after SCI is due to a signaling pathway that originates in the urothelium and then passes through gap junctions via a network of myofibroblasts in the lamina propria to smooth muscle and afferent nerves (Ikeda and Kanai, 2008). In our study, urodynamic assessment examined at 2 and 4 weeks after SCI supported the positive effect of MP and TIIA on recovery of bladder function. These include increases in voided volume and voiding efficiency and reductions in residual volume and in non-voiding contractions after SCI.
Electrophysiological studies in cats revealed that the recovery of bladder function after SCI is mediated by a change in the afferent limb of the micturition reflex pathway and remodeling of synaptic connections in the spinal cord. In chronic SCI cats, unmyelinated C-fiber afferents rather than Aδ afferents initiate voiding. These findings are supported by pharmacological studies showing that subcutaneous administration of capsaicin, a C-fiber neurotoxin, completely blocks reflex bladder contractions induced by bladder distention in chronic spinal cats; whereas capsaicin has no inhibitory effect on reflex bladder contractions in spinal intact cats (Yoshiyama et al., 1999;de Groat and Yoshimura, 2012). In this study, histological observation showed that bladder afferent neurons in the DRG (L 6 -S 1 ) undergo somal hypertrophy in SCI rats. However, that could be altered by TIIA or MP intervention, which also prevents bladder hypertrophy. Further research is needed to examine whether TIIA and MP might promote C-fiber bladder afferents to sprout and contribute to the synaptic remodeling of the spinal micturition reflex pathway after SCI.
The ionic mechanisms underlying the hyperexcitability of C-fiber bladder afferents were investigated using whole-cell patch clamp recording in bladder DRG neurons (Yoshimura and de Groat, 1997;Yoshiyama et al., 1999). Dissociated bladder DRG neurons from chronic SCI rats are larger in size and have increased input capacitance. This is consistent with results from histological studies showing that bladder afferent neurons in the DRG (L 6 -S 1 ) undergo somal hypertrophy in SCI rats (Kruse et al., 1995). The action potentials in bladder afferent neurons are also different after SCI in both rats and cats. In contrast to neurons from spinal intact rats where the majority (approximately 70%) of bladder afferent neurons exhibit high threshold tetrodotoxin-resistant action potentials (Yoshimura et al., 1996), in chronic SCI rats, 60% of bladder afferent neurons exhibit low threshold tetrodotoxin-sensitive action potentials. Therefore, it is likely that following SCI, A-type potassium channels are suppressed in parallel with an increased expression of tetrodotoxin-sensitive Na + currents, thereby increasing the excitability of C-fiber bladder afferent neurons. Thus, drugs that selectively modulate these types of ion channels might be useful in treating neurogenic detrusor overactivity. Whether TIIA or MP can modulate these types of ion channels would help establish the mechanism of their action.
In summary, TIIA could reduce the inflammation, edema as well as compensatory thickening of the bladder tissue and improve urodynamic parameters in adult rats at 2 and 4 weeks after SCI. Moreover, TIIA inhibited the increase of apoptotic and hypertrophic DRGs (L 6 -S 1 ) at 4 weeks after SCI. Taken together, TIIA could improve functional recovery in SCI-induced LUT dysfunction in adult rats and its effect is similar to MP. Although there were no dramatic differences between TIIA group and MP group, TIIA might be a better alternative for the treatment of SCI-induced LUT dysfunction. Large doses of MP application has caused many severe complications (Varma et al., 2013) so it is no longer recommended as the first-line drug for acute SCI (Walters et al., 2013). TIIA could be consecutive intravenously injected for 7 days but no side-effect.