Methods

Animals
The experimental protocol was approved by the local Ethical Committee for Animal Research (M38-13). Adult male Sprague-Dawley rats (n = 35) (Taconic Copenhagen, Denmark) were used to investigate the brain water content and the permeability-surface area product (PS) for [51Cr]-EDTA. There was a reversed 12–12 light cycle, with darkness during the day. The animals were treated in accordance with the national guidelines for laboratory animals (publications DFS 2004:4, the Swedish Board of Agriculture)
The animals were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg, Pentobarbitalnatrium, vet.APL, 60 mg/ml, APL, Stockholm) and then placed on a heating pad and in a stereotactic head holder. Body core temperature was kept at 37 °C by a feedback circuit controlled by continuous measurement of rectal temperature. A parasagittal craniotomy, with a diameter of 6 mm, was performed in the left parietal bone between the coronal and lambdoid sutures with the center approximately 5 mm from the coronal suture and 5 mm from the sagittal suture, after a scalp incision. The animals were randomly assigned to three different solutions applied to the surface of the exposed dura in a volume of 10 μL; i) vehicle (saline), ii) CFA used as stock solution (Sigma Aldrich, Life Science, US), and iii) an IS. The recipe of IS was adapted from Strassman and colleagues [29] and contains 10 μM each of bradykinin, serotonin and prostaglandin E2, 100 μM histamine at a pH of 5.0 (all obtained from Sigma Aldrich). After 15 min CFA or IS was removed by flushing with saline. Surgical staples were used to close the scalp incision and the animals were moved to another heating pad for recovering. At 2, 24 or 48 h following application to the dura, the animals were re-anesthetized. Anesthesia was induced by placing the rat in a covered glass-container with a continuous supply of isoflurane (Isofluran Baxter, Baxter Medical, Stockholm, Sweden). After tracheostomy, the rat was connected to a ventilator and anesthesia was maintained by inhalation of 1.4–1.7 % of isoflurane in room air. End tidal pCO2 was monitored continuously with a capnograph (CapnoTrue AMP, bluepoint MEDICAl, GmbH, Selmsdorf, Germany). The left femoral artery was canulated for measurement of mean arterial blood pressure (MAP) and to obtain arterial blood samples used for measurement of the physiological parameters pH, pCO2, pO2, hematocrit and electrolytes. The left femoral vein was canulated and used for infusions.

Tissue Water Content and the permeability-surface area product (PS) for [51Cr]-EDTA
Change in microvascular permeability and surface area for transvascular exchange following the various treatments was investigated by measurement of the permeability-surface area product (PS) for [51Cr]-EDTA, as described earlier [30, 31]. For this purpose the animals received a bolus infusion of about 370 kBq of the tracer [51Cr]-EDTA, (0.5 mL) (GE Health Care, Stockholm, Sweden). The bolus infusion was followed by a continuous infusion of the tracer at a rate of 0.33 mL/h (3.7 MBq/mL). Arterial blood samples (10 μL) for analyses of plasma [51Cr]-EDTA concentration were collected at 2.5, 5, 10, 15, 25, 35 and 40 min post start of the bolus injection. After 37 min, a bolus dose of about 25 kBq of [125I]-albumin dissolved in 0.1 mL isotonic saline was given in the femoral vein for calculation of plasma volume in the brain. Three min later, the experiment was finished by an arterial blood sample and the animal was sacrificed by decapitation. Before each experiment [125I]-albumin was purified from free iodine, using centrifugal filtration. The brain and the TGs were removed and put on a chilled support. In order to analyze PS in the cortex, periaqueductal gray (PAG), trigeminal nuclei caudalis (TNC) and cerebellum, tissue structures were extracted and immediately weighed. Tissue and blood tracer activities of 51 Cr-EDTA and 125I-albumin were determined in a gamma counter. Tissue samples were then dried in an oven for 24 h at 100 °C and brain water content was calculated as [(wet weight-dry weight)/wet weight)] × 100. Arterial hematocrit was measured before and during tracer infusion in order to convert blood concentrations into plasma concentrations. The blood to brain transfer constant (Ki) for [51Cr]-EDTA was then calculated according to the following equation Ki = B/ 0∫T Ca (t) d t [32], where B is the amount of tracer that has moved from blood to brain (tissue uptake of tracer minus regional tracer concentration in plasma), Ca is concentration of the tracer in arterial plasma as a function of time, and T is the duration of the experiment. Ki is a function of capillary plasma flow per unit mass of tissue (FV) and the permeability-surface area product (PS), the latter reflecting microvascular permeability and surface area available for diffusional exchange. The mathematical expression for this relationship is Ki = FV [1-e-PS/FV] which can be rewritten as PS = -FV ln (1-Ki/FV) [33]. From this expression it can be deduced that with a Ki/FV ratio of less than 0.1 the Ki value approximates PS with an error of less than 6 % [34].

Evans blue
The method has recently been published; briefly 2 % Evans blue in saline was injected in the tail vein (4 ml/kg) during anaesthesia (Eftekhari et al 2015). After 30 min the animal was sacrificed and the tissues dissected out. Evans blue couple to circulating albumin and forms a large complex (EBA) which does not pass the BBB at the resting state.

Photo microscopy
Sprague-Dawley rats (n = 3) were anesthetized and IS or CFA was added (see above) and monitored with a photo microscope (Olympus SZX10) at baseline and after 15 min following the addition of the inflammatory substances. For the three rats, parasagittal craniotomy, with a diameter of 6 mm, was performed and analyzed on both the left and right parietal bone.

Statistical analysis
All data are expressed as mean ± SE, where n equals individual animals. Data were analyzed statistically using Prism 6 (GraphPad Software, Inc, CA). Differences between groups were tested using non-parametric analyzes of variance with Kruskal-Wallis test followed by Dunn’s test for multiple comparisons. Each group was compared with the vehicle group.