FTIR Imaging of BrainTissue Reveals Crystalline CreatineDeposits Are an ex Vivo Marker of Localized Ischemia during MurineCerebral Malaria: General Implications for Disease Neurochemistry

摘要

Phosphocreatine is a major cellular source of high energy phosphates, which is crucial to maintain cell viability under conditions of impaired metabolic states, such as decreased oxygen and energy availability (i.e., ischemia). Many methods exist for the bulk analysis of phosphocreatine and its dephosphorylated product creatine; however, no method exists to image the distribution of creatine or phosphocreatine at the cellular level. In this study, Fourier transform infrared (FTIR) spectroscopic imaging has revealed the ex vivo development of creatine microdeposits in situ in the brain region most affected by the disease, the cerebellum of cerebral malaria (CM) diseased mice; however, such deposits were also observed at significantly lower levels in the brains of control mice and mice with severe malaria. In addition, the number of deposits was observed to increase in a time-dependent manner during dehydration post tissue cutting. This challenges the hypotheses in recent reports of FTIR spectroscopic imaging where creatine microdeposits found in situ within thin sections from epileptic, Alzheimer’s (AD),and amlyoid lateral sclerosis (ALS) diseased brains were proposedto be disease specific markers and/or postulated to contribute tothe brain pathogenesis. As such, a detailed investigation was undertaken,which has established that the creatine microdeposits exist as thehighly soluble HCl salt or zwitterion and are an ex-vivo tissue processingartifact and, hence, have no effect on disease pathogenesis. Theyoccur as a result of creatine crystallization during dehydration (i.e.,air-drying) of thin sections of brain tissue. As ischemia and decreasedaerobic (oxidative metabolism) are common to many brain disorders,regions of elevated creatine-to-phosphocreatine ratio are likely topromote crystal formation during tissue dehydration (due to the lowerwater solubility of creatine relative to phosphocreatine). The resultsof this study have demonstrated that although the deposits do notoccur in vivo, and do not directly play any role in disease pathogenesis,increased levels of creatine deposits within air-dried tissue sectionsserve as a highly valuable marker for the identification of tissueregions with an altered metabolic status. In this study, the locationof crystalline creatine deposits were used to identify whether analtered metabolic state exists within the molecular and granular layersof the cerebellum during CM, which complements the recent discoveryof decreased oxygen availability in the brain during this disease.