A method to image brain tissue frozen at autopsy.

Magnetic Resonance Imaging (MRI) can provide the location and signal characteristics of pathological regions within a postmortem tissue block, thereby improving the efficiency of histopathological studies. However, such postmortem-MRI guided histopathological studies have so far only been performed on fixed samples as imaging tissue frozen at the time of extraction, while preserving its integrity, is significantly more challenging. Here we describe the development of cold-postmortem-MRI, which can preserve tissue integrity and help target techniques such as transcriptomics. As a first step, RNA integrity number (RIN) was used to determine the rate of tissue biomolecular degradation in mouse brains placed at various temperatures between -20 °C and +20 °C for up to 24 h. Then, human tissue frozen at the time of autopsy was immersed in 2-methylbutane, sealed in a bio-safe tissue chamber, and cooled in the MRI using a recirculating chiller to determine MRI signal characteristics. The optimal imaging temperature, which did not show significant RIN deterioration for over 12 h, at the same time giving robust MRI signal and contrast between brain tissue types was deemed to be -7 °C. Finally, MRI was performed on human tissue blocks at this optimal imaging temperatures using a magnetization-prepared rapid gradient echo (MPRAGE, isotropic resolution between 0.3-0.4 mm) revealing good gray-white matter contrast and revealing subpial, subcortical, and deep white matter lesions. RINs measured before and after imaging revealed no significant changes (n = 3, p = 0.18, paired t-test). In addition to improving efficiency of downstream processes, imaging tissue at sub-zero temperatures may also improve our understanding of compartment specificity of MRI signal.

Switching a HO···π Interaction to a Nonconventional OH···π Hydrogen Bond: A Completed Crystallographic Puzzle.

In this article, we present crystallographic and spectroscopic evidence of a tunable system wherein a HO···π interaction switches incrementally to a nonconventional OH···π hydrogen bonding (HB) interaction. More specifically, we report the synthesis of substituted forms of model system 1 to study the effects of aryl ring electronic density on the qualitative characteristics of OH···π hydrogen bonds therein. The OH stretch in experimental infrared data, in agreement with density-functional theory (DFT) calculations, shows continuous red-shifts as the adjacent ring becomes more electron rich. For example, the OH stretch of an amino-substituted analogue is red-shifted by roughly 50 cm-1 compared to the same stretch in the CF3 analogue, indicating a significantly stronger HB interaction in the former. Moreover, DFT calculations (ωB97XD/6-311+G**) predict that increasing electronic density on the adjacent top ring reduces the aryl π-OH σ* energy gap with a concomitant enhancement of the OH n-π* energy gap. Consequently, a dominant π-σ* interaction in the amino substituted analogue locks the system in the in-form while a favorable n-π* interaction reverses the orientation of the oxygen-bound hydrogen in its protonated form. Additionally, the 1H NMR data of various analogues reveal that stronger OH···π interactions in systems with electron-rich aromatic rings slow exchange of the alcoholic proton, thereby revealing coupling with the geminal proton. Finally, X-ray crystallographic analyses of a spectrum of analogues clearly visualize the three distinct stages of "switch"-starting with exclusive HO···π, to partitioned HO···π/OH···π, and finally to achieving exclusive OH···π forms. Furthermore, the crystal structure of the amino analogue reveals an interesting feature in which an extended HB network, involving two conventional (NH···O) and two nonconventional (OH···π) HBs, dimerizes and anchors the molecule in the unit cell.

Postmortem MRI of Tissue Frozen at Autopsy.

Introduction: Postmortem MRI provides insight into location of pathology within tissue blocks, enabling efficient targeting of histopathological studies. While postmortem imaging of fixed tissue is gaining popularity, imaging tissue frozen at the time of extraction is significantly more challenging. Methods: Tissue integrity was examined using RNA integrity number (RIN), in mouse brains placed between -20 °C and 20 °C for up to 24 hours, to determine the highest temperature that could potentially be used for imaging without tissue degeneration. Human tissue frozen at the time of autopsy was sealed in a tissue chamber filled with 2-methylbutane to prevent contamination of the MRI components. The tissue was cooled to a range of temperatures in a 9.4T MRI using a recirculating aqueous ethylene glycol solution. MRI was performed using a magnetization-prepared rapid gradient echo (MPRAGE) sequence with inversion time of 1400 ms to null the signal from 2-methylbutane bath, isotropic resolution between 0.3-0.4 mm, and scan time of about 4 hours was used to study the anatomical details of the tissue block. Results and Discussion: A temperature of -7 °C was chosen for imaging as it was below the highest temperature that did not show significant RIN deterioration for over 12 hours, at the same time gave robust imaging signal and contrast between brain tissue types. Imaging performed on various human tissue blocks revealed good gray-white matter contrast and revealing subpial, subcortical, and deep white matter lesions typical of multiple sclerosis enabling further spatially targeted studies. Conclusion: Here, we describe a new method to image cold tissue, while maintaining tissue integrity and biosafety during scanning. In addition to improving efficiency of downstream processes, imaging tissue at sub-zero temperatures may also improve our understanding of compartment specificity of MRI signal.