Biology subjects

German, A.

Publications and source records attributed to German, A..

4 recordsLinked to original sources

bioRxiv2026

Ultrastructural comparison of fixation and cryopreservation methods for brain preservation

Maximizing the morphological and molecular fidelity of preserved mammalian brain tissue is essential for basic neuroscience and brain banking, where tissue quality determines the reliability of downstream analyses. Aldehyde fixation and cryogenic storage are the most powerful preservation techniques available. However, how to best combine them is incompletely characterized. Here, we tested twelve different approaches to preserve the murine brain, including standard aldehyde perfusion fixation, variants of aldehyde-stabilized cryopreservation (ASC) using ethylene glycol (EG), aldehyde fixation followed by unprotected freezing, and several cryopreservation protocols without preceding aldehyde fixation, such as interleaved equilibration with vitrification solution. Preservation quality was assessed using light microscopy, transmission electron microscopy, and patch-level feature analysis with the DINOv2 vision foundation model. We found that ASC with sodium dodecyl sulfate (SDS)-mediated blood-brain barrier permeabilization preserved nuclear morphology, myelin periodicity, and neuropil texture comparable to standard aldehyde perfusion fixation, providing the first independent replication of ASC. Omitting SDS caused severe parenchymal dehydration, due to a mismatch between water and cryoprotectant transport. Aldehyde fixation followed by unprotected slow freezing confined ice damage primarily to perivascular zones, while fast freezing led to intranuclear clefts and cavities. Among non-fixation protocols, interleaved equilibration with vitrification solution preserved myelinated axon profiles and chromatin patterns, but induced perivascular edema. High concentration glycerol perfusion caused significant osmotic dehydration. Straight freezing of unfixed tissue without cryoprotectant was followed by membrane disruption, but resulted in the visualization of more structure than expected, likely as a result of structural restitution upon thawing. Taken together, our results provide a framework for matching preservation strategies to the needs of different types of brain banking and neuroscience research.

neuroscience↗

bioRxiv2025

CRYO-CEST: Non-invasive imaging of cryoprotectants using chemical exchange saturation transfer

Chemical exchange saturation transfer (CEST) MRI at 7T was explored as a non-invasive method to detect and quantify common cryoprotectants used in organ cryopreservation. Phantom experiments demonstrated clear CEST signals from ethylene glycol, formamide, and dimethyl sulfoxide, with formamide showing the most sensitive concentration-dependent response. CEST imaging could thus provide a practical tool for monitoring cryoprotectant concentrations in organs.

biophysics↗

bioRxiv2025

Functional recovery of adult brain tissue arrested in time during cryopreservation by vitrification

Cryopreserving the adult brain is challenging due to damage from ice formation, and traditional freezing methods fail to maintain neural architecture and function. Vitrification offers a promising alternative but has not been surveyed in the brain. Here, we demonstrate near-physiological recovery of the adult murine hippocampus after vitrification of brain slices and of the whole brain in situ. Key features of the hippocampus are preserved, including structural integrity, metabolic responsiveness, neuronal excitability, and synaptic transmission and plasticity. Notably, hippocampal long-term potentiation was well preserved, indicating that the cellular machinery of learning and memory remains operational. These findings extend known biophysical limits for cerebral hypothermic shutdown by demonstrating recovery after complete cessation of molecular mobility in the vitreous state. This suggests that the brain can be arrested in time and then reactivated, opening avenues for potential clinical applications. Significance StatementWhile the brain is considered exceptionally sensitive, we show that the hippocampus can resume normal electrophysiological activity after being rendered completely immobile in a cryogenic glass. The work extends known biophysical tolerance limits for the brain from the hypothermic to the cryogenic range and establishes a protocol for its long-term storage in a viable state.

neuroscience↗

bioRxiv2024

Excitability and synaptic transmission after vitrification of mouse corticohippocampal slices.

Cryopreservation of adult neural tissue is of considerable practical and theoretical interest. Utilizing 61% w/v ethylene glycol, we vitrified and rewarmed acute mouse corticohippocampal slices to evaluate field excitatory postsynaptic potentials (fEPSP) in the stratum radiatum of the CA1 region of the hippocampus. Our results demonstrate successfully recovered synaptic transmission, and high-frequency stimulation (HFS)-induced potentiation. However, we failed to induce a stable potentiation following HFS stimulation. Structural analysis post-vitrification revealed cellular alterations such as swelling and vacuolization, which likely contributed to the unstable potentiation. Despite high variability in results, this study highlights the potential of vitrification to partially preserve brain function.

neuroscience↗