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Abstract: Hypothermic storage of red blood cell is essential in the field of transfusion medicine to fulfill the huge clinical demand for red blood cell products in situations of massive bleeding or red blood cell disorders. The prime goal of hypothermic storage is to maintain the quality and safety of stored red blood cells. The cell membrane is one of the red blood cell main components that needs to be structurally and functionally preserved throughout storage. It is characterized by its semipermeability which allows the movement of water to maintain osmotic equilibrium with external media and allows for cell shrinkage or swelling. The cell membrane, however, experiences progressive damages during storage. Several factors are proposed to influence the storage lesion including the storage length, blood component manufacturing method, and donor characteristics. This thesis will characterize the membrane water permeability properties during storage and will apply water permeability measurements to determine the contributions of the storage length, blood component manufacturing method, and donor-related characteristics to membrane water permeability variation. Red blood cell units were obtained from volunteer blood donors and categorized according to the duration of storage, manufacturing method, and donor characteristics. Validated techniques were applied to characterize the red cell membrane water permeability and membrane quality properties. The membrane water permeability parameters included the hydraulic conductivity of water permeability, osmotically inactive fraction, and Arrhenius activation energy. The membrane quality measures were deformability, total hemoglobin, supernatant hemoglobin, percent hemolysis, osmotic fragility, hematologic indices of mean corpuscular iii volume, the mean corpuscular hemoglobin, and the mean corpuscular hemoglobin concentration, supernatant potassium, and supernatant sodium. It was found that membrane water permeability measurements were significantly increased as a function of the storage length (at day 21 of storage), which remained elevated for the rest of 42 days of storage. This was accompanied by significant elevations in the osmotically inactive fraction and Arrhenius activation energy. This study, also, found that units processed by a whole blood filtration manufacturing method exhibited significantly higher membrane water permeability from the start of storage until the end of 42 days of storage compared to units manufactured using a red cell filtration. There were no significance differences between groups in dependent to donors’ age and sex. These results revealed that both the storage length and manufacturing process had significant contributions to the water permeability and various quality parameters with limited contributions from the blood donors’ age and sex. In conclusion, this thesis has expanded the knowledge of membrane quality during the hypothermic storage by applying a novel method to assess membrane water permeability. According to these findings, it is therefore likely that such connections exist between water permeability increase and membrane damages during storage. This work also tested possible factors affecting the RBCs quality during hypothermic storage. Findings of this thesis proposed that part of the membrane-related storage lesion occur before the first day of storage due to the effect of blood manufacturing process and donor variability. The findings of this thesis highlight the importance of describing membrane water permeability properties during storage which can be a quality predictor of the biophysical and chemical changes that affect the quality of stored red blood cells during hypothermic storage.

Osmotic brain edema or chronic treatment with desipramine alters brain water permeability. In this study we investigated aquaporin 4 expression and distribution in these two conditions. Brain edema development was induced by intraperitoneal water injection. Blood serum osmolality decreased from 296 ± 1 mOsm to 278 ± 2 mOsm within 15 min. Cerebral cortex water content increased from 79.8 ± 0.2 % to 81.3 ± 0.5% during 120 min of this hyposmotic exposure. Aquaporin 4 immunostaining intensity at the astrocytic endfeet increased in water injected animals from 2.6 ± 0.04 intensity unites (IU) to 3.2 ± 0.21 IU, while total brain AQP4 expression remained unaltered. Chronic treatment with desipramine showed no significant change in serum osmolality or cerebral cortex water content. Similar to water injected animals, aquaporin 4 immunostaining intensity in chronic desipramine animals increased at the astrocytic endfeet (2.5 ± 0.04 IU to 3.0 ± 0.13) but total cortical AQP4 expression was again unaltered. These experiments suggest that decreased brain water permeability caused by osmotic brain edema is not a result of decreased AQP4 expression or localization of astroglial endfeet. However chronic treatment with desipramine increases AQP4 immunostaining at the astrocytic endfeet without increasing overall AQP4 expression.

Water transport has been widely studied in a variety of cells and isolated membranes. However, the intrinsic complexity related with the study of intracellular processes has delayed the development of an analytical technique for the study of water transport in subcellular compartments of living cells. This study represents the development and application of a ratiometric fluorescent probe to study water transport in endocytic organelles of intact living cells. The probe is based on the use of a D2O-sensitive fluorophore, Lucifer yellow dextran (LY-dex), together with a D2O-insensitive fluorophore, Alexa fluor 546 dextran (AF546-dex), used as an internal reference. To ensure localization of the dyes into the organelles via pinocytocis and avoid leaking, both dyes are coupled to a high molecular weight (10.000MW) aminodextran In the first part of the thesis, demonstration of the efficacy of the probe to measure water transport in living cells is shown. In the second part, the probe is used for quantification of water exchange in lysosomes. Finally, the probe is employed to follow kinetics of water exchange in macropinosomes and lysosomes. It was demonstrated that the probe responds linearly to changes in D2O concentration (% v/v). Inin vitro experiments the probe ratios changed about 0.40 units between 0%v/v and 90% v/v D2O, while in ex vivo experiments the probe ratio changes about 0.20 units over the same concentration range. To probe its applicability, studies of superfusion were conducted with Ringer's Buffer containing D2O over the cells incubating in buffer prepared in H2O. Rapid imaging of the organelles during perfusion experiments allowed the determination of kinetics of water exchange across the membranes of lysosomes in CHO cells. The probe was also used in the quantification of water exchange in J774.A1 lysosomes. It was observed that J774.A1 lysosomes exchange 67 -41 % of their water content under equilibrium but when the organelles were permeabilized, using 20 microg/ml digitonin solutions in 90% D2O to create an open system, lysosomes exchange the remaining water content. Taking advantage of the fact that organelles loaded with the fluorescent probe show significant increases in LY-dex fluorescence intensity as D2O from the extracellular solution is exchanged across the organellar membrane, the probe was used to follow the kinetics of water exchange across the membranes of lysosomes and macropinosomes in J774.A1 cells. It was found that lysosomal water permeability is pH dependent; changing from (6.8 0.2) x 10-3cm/s (mean s.e.m. n=62 cells) to (9.4± 0.5) x 10-4 (mean s.e.m. n=22 cells) following treatment with NH4Cl and (1.3 ± 0.4) x 10-4 (mean s.e.m. n=4 cells) after treatment with Bafilomycin A1. It was also observed that macropinosome membrane permeability increases from (1.2 ±0.6) x 10-3 cm/s (3 min after formation mean s.e.m. n=15 cells) to (5.2 ±0.6) x 10-3 cm/s (after 25 min of formation mean s.e.m. n=15 cells). iv Collectively these findings demonstrate the ability of the developed methods for quantification and determination of the kinetics of water exchange in subcellular organelles in living cells. This is the first time that kinetics of water and quantification of water has been measured in organelles under physiological conditions.