Interstitial Fluid

The bacteriophage sensitive strains used during the production and quality control of BFC-1 are P. aeruginosa strain ‘573’, were isolated at the Eliava Institute of Bacteriophage, Microbiology and Virology (EIBMV) in the 1970s from bone marrow interstitial fluid, and S. aureus strain '13 S44 S′, isolated at the Brussels Burn Centre in 2006 from a burn wound. Initially, S. aureus strain Wood 60 (EIBMV collection) was used for propagation of phage ISP, but for the production of this cocktail the phage was propagated on S. aureus 13 S44 S. The absence of temperate phages from the host strains was tested as described in a separate section of this paper.
For bacteriophage isolation from natural samples such as sewage and river water, one millilitre of 10×concentrated LB Broth (Becton Dickinson), 1 ml ‘host bacteria’ suspension, containing 10⁸ cfu in LB broth and 9 ml sewage or river water were mixed in a 14 ml sterile tube. This tube was incubated at 37°C for 1.5–2 h. Subsequently, 200 µl of chloroform (Sigma-Aldrich, Bornem, Belgium) was added and the tube was further incubated at 4°C for 1 h. The lysate was aspirated with a sterile 5 ml syringe and passed through a 0.45 µm membrane filter (Minisart, Sartorius, Vilvoorde, Belgium). Bacteriophages were titrated using the agar overlay method, as described above. All plaques with different morphology were touched with a sterile pipette tip, inoculated into 2 ml of sterile LB broth in 14 ml sterile tubes and incubated at 37°C for 2 h. Subsequently, 50 µl of chloroform was added and the tube(s) were incubated at 4°C for 1 h. For each tube, a dilution series (log(0)−log(−4)) was made in sterile 14 ml tubes filled with LB broth. Each dilution was titrated using the agar overlay method. Plates showing 1–10 plaques were analysed in detail. Again, all plaques with different morphology were touched with a sterile pipette tip, inoculated into 2 ml of sterile LB broth in 14 ml sterile tubes and incubated at 37°C for 2 h. This complete cycle was repeated until one plaque morphotype was obtained (homogeneous plaques).
In the case of bacteriophage ISP, which was isolated in the 1920s, porcelain, rather than membrane filters were employed.

Prior to normalization using the diffeomorphic DARTEL approach, the MRI data were segmented into different tissue classes. Image processing was done using SPM8 release 4010 (Wellcome Trust Centre for Neuroimaging, London, UK) using the AA version 3.01 pipeline (http://www.cambridgeneuroimaging.com/aawiki/). Each individual's structural image was first coregistered to an ICBM152-space (i.e., MNI-space) average template distributed with SPM8 using normalized mutual information. This ensured reasonable starting estimates for the unified segmentation routine, and was done as an alternative to manually repositioning each scan. Structural images were then segmented into tissue classes using unified segmentation (Ashburner and Friston, 2005 (link)) as implemented in the “new segment” option of SPM8. This segmentation makes use of a number of tissue probability maps including GM, white matter (WM), cerebrospinal fluid (CSF), soft tissue, skull, and non-brain regions of the image. These maps reflect the prior probability of a given voxel belonging to a tissue class based on a large sample of healthy adults across the lifespan (Good et al., 2001 (link)). This information, in combination with the distribution of voxel intensities, is used to assign a probability to each voxel of belonging to a particular tissue class using Gaussian mixture modeling. Unless otherwise specified, default values were used for segmentation, except that the data were sampled every 1 mm (instead of the default 3 mm).
We also performed a second, parallel analysis using the alternative “standard” unified segmentation from SPM5/SPM8 (that has been used in many previous VBM studies). This segmentation uses fewer tissue classes (GM, WM, CSF), and tissue probability maps are based on a sample of young adults only (and hence potentially biased when examining age effects). Although not discussed at length in the main text, quite different effects of age were obtained from this “standard” segmentation analysis (shown in Supplemental Fig. S1–S3) relative to the “new” segmentation analysis reported in the main text.
Prior to segmentation, bias-corrected structural images were created to reduce the influence of intensity inhomogeneity on segmentation; producing a separate bias-corrected image effectively results in a two-pass bias correction, as bias correction is built-in to the segmentation process. Additionally, to reduce the likelihood that non-brain voxels were classified as GM, WM, or CSF, the tissue probability maps for these three tissue classes were set to 0 outside of a template-space brain mask.3 Following segmentation, images for each tissue class were roughly registered in a common space using a rigid body transformation. Segmented images were written out at 1.5 mm isotropic resolution.
The volume of the resulting GM, WM, and CSF tissue classes was determined from the (unsmoothed, unregistered) segmented images by integrating over all voxels and multiplying by voxel size, and the volumes of these three classes were summed to provide an estimate of total intracranial volume (TIV).

CGM measures glucose in interstitial fluid through subcutaneous sensor and saves data in the recorder every 5 minutes. It has been validated by several studies and was also known to provide a very well correlation between blood and interstitial fluid glucose values.^{[17 (link)]} A minimum of 3 self-monitoring of blood glucose values per day from glucometer are needed to calibrate the glucose sensor data. CGM measurements provided several important information of GV, including time per day within target glucose range (time-in-range, glucose levels between 70–180 mg/dL), time-above target glucose range (time-above-range, glucose levels > 180 mg/dL), and time-below target glucose range (Time-Below-Range, glucose levels < 70 mg/dL). GV indices, which included standard deviation (SD), (magnitude of glycemic excursions [MAGE], average of blood glucose excursions exceeding 1 SD of the mean blood glucose value), (mean of daily differences [MODD], the absolute difference between the paired CGMS values obtained during 2 successive days (minimum and maximum SD days), and (continuous overall net glycemic action [CONGA], SD of differences between observed blood glucose reading and an observed blood glucose level).^{[18 (link),19 (link)]}

To verify whether ADC and D may reflect the volume of interstitial fluid (ISF) space in the brain parenchyma, we performed an additional DWI measurements by mimicking increasing ISF space volume in 3 water phantoms filled with Sephadex G-25 microbeads (Sephadex G-25; Sigma-Aldrich, St. Louis, MO, USA) of coarse (100–300 μm), medium (50–150 μm), fine particle size (20–80 μm of wet particle size). All microbeads possess the same porosity <5 kD so a similar exchange rate between stored and free water pools is expected. Each phantom was of the same in-house design (in. volume ~0.5 ml.), formed from of thick plexiglas tube (~25 mm in. long, out./in. ø10/5 mm) with a thread plug at both sides. The thread plug was made from the same tube, with a standard bonded polyester microfilter placed inside (Appendix 1—figure 1E) to prevent evacuation of the microbeads. For DWI, microbeads for each phantom were placed initially in a distilled water for 24 hr, to achieve their maximal size. Afterwards, a 2 ml syringe filled with distilled water solution of 0.001 mM/ml gadobutrol was attached to the thread plug on one side of the phantom and the phantom was filled with the microbeads. Gadobutrol was used to obtain optimal MR signal shortening for the echo time used in the same EPI sequence as employed in vivo (Table 1B). Any possible air bubbles remaining between the microbeads were carefully removed using a 1 µl inoculating loop. Subsequently, the other side of the phantom was closed with the thread plug, the phantom was flushed with ~1.8 ml gadobutrol solution in the attached syringe, and the solitary plug was sealed with a rubber syringe cap and parafilm. The syringe with a residual of gadobutrol solution at the other end was left to support pressure equalization inside the phantom.
DWI was performed 6 times in each phantom, with a central slice imaged in 1/3 phantom’s distal portion from the syringe. To verify the diffusion values in a free water environment, DWI was performed 4 times in the same phantom filled solely with gadobutrol solution.