Iontophoresis

C57BL/6J male mice at age P56 were injected with EGFP-expressing AAV using iontophoresis by the stereotaxic method. The brains were scanned using the STP tomography systems. Images were subject to data quality control and all the injection sites were manually annotated. All the image sets were co-registered into the 3D reference space. EGFP-positive signals were segmented from background, and binned at voxel levels for quantitative analyses. The raw connectivity data are served with various navigation tools on the web through the Allen Institute’s data portal. See the full Methods section for detailed descriptions.

The concept underlying methods of direct diffusion measurements is to introduce a detectable substance into the ECS and to subsequently measure its concentration distribution in space and time. In an ideal situation this distribution will be described by an appropriate solution to Equation 9 and, by fitting this solution to the measured concentration distribution, the parameters λ and α may be obtained. When there are other processes, such as loss characterized by a first-order kinetic constant k′, then the additional parameters may be extracted also. An important, but sometimes neglected, caveat is that the experimental concentration distribution must be accurately represented by the mathematical solution to the diffusion equation; otherwise, derived parameter values are meaningless. Therefore it is essential that a distribution curve can be obtained by the method of measurement, rather than a single value, to compare with theoretical predictions.
The choice of probe substance is important; the probe should be small enough to explore all regions of the ECS but remain predominantly in the ECS compartment. The probe should be non-toxic to brain tissue and of sufficiently low concentration that it does not affect interstitial osmolarity. Finally, it must be possible to measure the substance with adequate selectivity and sensitivity over endogenous compounds to determine a range of concentrations. These are quite stringent constraints. Once measurements with an ideal probe are available, the behavior can be compared with, for example, the non-ideal behavior of a drug that is taken up by cells or removed across the BBB to determine information about the elimination of the drug. Use of a larger probe compound that is more impeded in its diffusion by the narrow interstitial spaces of the ECS may provide additional information, such as an estimate of the size of the spaces.
The following sections will consider the older radiotracer method, the real-time iontophoresis (RTI) and real-time pressure (RTP) ejection methods, and the integrative optical imaging (IOI) method. After describing the techniques, the major results obtained with these methods will be reviewed with the exception of the large body of results obtained with the RTI method employing the cation tetramethylammonium (TMA⁺). The RTI-TMA data will be treated separately because this extensive and diverse collection of experiments forms the basis for much of our present understanding of the diffusion properties of the ECS and it has been the predominant technique for exploring the changed diffusion properties in development, aging, various forms of experimental intervention, and pathological states.

BONUS was conducted at 28 US Cystic Fibrosis Foundation–accredited Care Centers in the CF Foundation Therapeutic Development Network. Infants younger than 3.5 months were enrolled if they had a sweat chloride level of at least 60 mEq/L (to convert to millimoles per liter, multiply by 1) by using a quantitative pilocarpine iontophoresis test or 2 well-characterized CF transmembrane conductance regulator gene (CFTR [NCBI Entrez Gene 1080]) mutations. Infants with a gestational age of less than 35 weeks, with a birth weight of less than 2.5 kg, unable to take full oral feeds, or with any serious condition other than pancreatic insufficiency (PI) contributing to malabsorption or interfering with normal growth were excluded. Study visits coincided with guideline-recommended CF clinic visits (monthly until 6 months and at 8, 10, and 12 months of age).^{7 (link)} Anthropometric measures (length, weight, and occipital frontal circumference) were systematically performed at each visit by certified staff.^{11 (link)} Participant guardians completed 3-day diet, stool,^{12 (link)} modified pain,^{13 (link)} and cough^{14 (link)} diaries for the infant before each study visit (eMethods 1 in the Supplement). Breastfeeding frequency and duration, salt intake, vitamin and formula supplementation, and PERT dosages were included in the diet diary. Caloric and nutritional analyses were performed, and the mean was calculated for the 3-day collection. Measurement of fecal elastase levels, respiratory tract cultures, and chest radiography were performed according to Cystic Fibrosis Foundation care guidelines,^{7 (link)} and the 12-month chest radiograph was scored^{15 (link)} centrally by 2 independent readers. Clinical hematologic levels, chemistry, and serum vitamin levels (A, D, and E) were collected at enrollment and 6 and 12 months. Additional details can be found in eMethods 2 in the Supplement. A complete list of cites is found in the list of BONUS investigators at the end of the article. Written informed consent was obtained from all participating parents or guardians, and all participating sites received approval from their institutional review boards.