The Sole Effect of Hypothermia on Blood-Brain Barrier Tight Junction Protein Expression
AdvisorRonaldson, Patrick T.
MetadataShow full item record
PublisherThe University of Arizona.
RightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
AbstractWhile the use of hypothermia as an organ preservation technique has been around since the mid-20th century, its use in combination with cardiopulmonary bypass (CPB) did not gain popularity until 1975 when it was demonstrated to offer a practical and safe approach for aortic arch surgery2,4. It has since been the cornerstone of successful cerebral protection during complex cardiovascular procedures4. The use of deep hypothermia (<28°C) in deep hypothermic circulatory arrest (DHCA) procedures can prevent cerebral ischemic injury through reductions in both anaerobic glycolysis and uncontrolled excitatory neurotransmitter release, which normally cause cell damage and eventual death5,6,7. Although these neuroprotective effects are well understood, neurological dysfunction is still the number one cause of morbidity and mortality following DHCA procedures, highlighting the need for improvements to increase cerebral protection20. The blood-brain barrier (BBB) provides precise regulation of CNS uptake of specific solutes required for maintenance of cerebral homeostasis while maintaining protection from harmful substrates that circulate in the blood. Tight junction (TJ) proteins, found between brain microvascular endothelial cells, significantly limit paracellular diffusion and prevent free exchange of compounds between the parenchyma and blood25. Indeed, TJ protein complexes are critical in providing a “physical barrier” to solute permeation at the level of the cerebral microvasculature. Modulation of TJ protein complexes, via changes in expression or trafficking of constituent proteins, can cause increased BBB permeability (i.e., leak). The consequences of BBB leak include an increased ability of potentially toxic substances to accumulate in brain parenchyma and cause significant cellular damage to neurons25. In fact, changes in BBB TJ protein expression during deep hypothermic conditions alone has not yet been investigated. This highlights the need for a clinically translational model that can be used to study TJ protein expression during these drastic changes in body temperature. The purpose of this study was to develop a clinically relevant rodent model for inducing deep hypothermia and to investigate changes in expression of transmembrane TJ proteins occludin and claudin-5. Animals were assigned to either control (CTRL), anesthesia (AN), or anesthesia and ice (AN+ICE) groups. Desired rectal temperature ranges were set at 33-31°C for AN group and 26-24°C for AN+ICE group for the Pilot Experiment and Experiment #1. AN group temperature range was adjusted to 37-35°C for experiment #2. Improvements to the model included: tighter control of active cooling the AN+ICE group, increased time within the assigned temperature ranges, implementation of EKG monitoring, control of respiration through ventilator use, verification of ventilator settings through arterial blood gas monitoring, and the use of ketamine/isoflurane combination as an anesthetic. Changes in expression of occludin and claudin-5 were evaluated via western blotting and subsequent densitometric analysis. Results of western blot experiments showed significant decreases in occludin in both AN (p<0.05) and AN+ICE (P<0.01) groups when compared to CTRL following the pilot experiment. The significant decrease (p<0.05) in the AN+ICE group when compared to CTRL was observed following Experiment #1 and #2. The AN group showed no significant differences compared to CTRL following Experiment #1 and #2. Significant differences in claudin-5 expression were not detected following the Pilot Experiment and Experiment #1. A significant decrease in claudin-5 expression was observed in the AN+ICE group when compared to the AN group following Experiment #2. In summary, we have successfully developed a rodent model of deep hypothermia that can be used for the in vivo study of brain microvascular changes that occur in response to clinically relevant changes in body temperature. Specifically, we have utilized this model to show that decreased body temperature causes measurable changes in occludin expression at the BBB. These observations form the basis for a more detailed examination of BBB dysfunction following deep hypothermia in an effort to understand how BBB injury relates to neurological complications following DHCA.
Degree ProgramGraduate College