Currently Funded Projects
Histological and anatomical preservation of rat brains after vitrification and rewarming
Conducted by Dr. Rodolfo Goya (National University of La Plata, La Plata City, Argentina)
The aim of the project is to assess the degree of histologic and anatomical preservation of rat brains after pre-mortem perfusion with a vitrification protocol used in human patients, scaled down for young rats, followed by a 3-stage cooling protocol that ends in liquid nitrogen. This protocol will be followed, between 2 and 7 days later, by rewarming and standard fixation, sectioning, and Nissl and immunohistochemical staining of relevant brain regions. Young adult Sprague–Dawley female rats will be placed under deep anesthesia and intracardially perfused with three cryopreservation solutions progressively enriched in ethylene glycol (EG), which will range from 10 to 70% EG. The third solution (termed VM1) includes 15% dimethyl sulfoxide (DMSO) as a permeabilizing agent. Different experimental groups of 4-5 rats each will be used. A control group which will be perfused with fixative (paraformaldehyde 4% in buffered saline) and not frozen (standard procedure) and the vitrified groups, which will be perfused with the vitrification solutions and frozen as indicated. Comparisons will be made for the different parameters under study in the different experimental groups, which will be perfused immediately or 90 minutes after death. This should clarify the impact of the length of time between death and vitrification on the preservation of brain structural integrity.
You can read the current progress report here.
Histological characterization of VM-1 cryoprotected brains
Conducted by Advanced Neural Biosciences, Inc. for the Hirsch Foundation 2017 – 2018
Cooling complex tissues to ultra-low temperatures produces severe damage, as measured by cell viability tests and microscopy. To prevent this kind of damage, so called “cryoprotectants” are used to minimize ice formation. Vitrification agents use very high concentrations of cryoprotectant to completely inhibit ice formation. VM-1 is a vitrification agent that is optimized for brain cryopreservation. Isolated brain slices can be exposed to VM-1 and recover viability after cooling to cryogenic temperatures. VM-1 also permits ice-free cryopreservation of the whole brain if the agent is introduced through the circulatory system of a mammal. The objective of this research project is to optimize introduction and removal of VM-1 and preserve the fine structure of the brain. First, we compared images of normal brains against brains cooled to 5 degrees Celsius. Then we compared different chemical preservation protocols to prepare cryoprotected brains for electron microscopy. After optimizing a chemical preservation protocol, we compared several protocols to introduce and remove VM-1 from the brain. The best outcome was observed when the brain was chemically prepared for microscopy before removal of the cryoprotectant. This protocol still showed evidence of severe brain shrinking. Further testing and optimization of VM-1 cryoprotection protocols is desired to eliminate this phenomenon.
You can find further information and the full scientific report here.