As a result of some of the research of Dr.Yuri Pichugin, the Cryonics Institute's Director of Research, changes have been made in the washout solution and the perfusate vehicle solution. Following are the compositions of the new solutions, which yield significantly better results than the previous solutions.
In addition, there had been discussion, in-house and with various consultants, over the merits of solutions or ingredients used or recommended by others. In particular some had recommended use of HES instead of mannitol in the washout solution, and some had recommended use of organ-preservation solutions such as Viaspan and its successors and competitors, including a solution known as m-RPS-2, a modified renal preservation solution, which differs from the new CI vehicle solution in that it contains glutathione. Direct comparisons were made, as summarized below, and the suggested alternatives were found not to be better than the new solutions now adopted.
The following is my summary, and not Dr. Pichugin's full report, but I think I have avoided errors and included the essentials.
A. The new CI washout solution:
|
mM/L |
Molecular Weight |
grams/L |
KCl
NaHCO3
KH2PO4
MgCl2·6H2O
Glucose
Mannitol
|
30.0
10.0
7.2
2.0
180.0
50.0
|
74.55
84.01
136.1
246.48
180.20
182.17
|
2.24
0.24
0.98
0.49
32.44
9.11
|
To adjust pH of the solution to 7.6,dissolve1g KOH in 2 ml of distilled water and add 15 drops of this to each liter of solution.
B. The new CI perfusate vehicle solution (to which glycerol is added in phased steps):
Same as above except that there is no mannitol.
C. Comparison of new CI washout solution with one using HES (50 g/L) instead of mannitol:
Rat brain hippocampal slices were incubated at 29oC for one hour before use. Slices were then kept in the CI or comparison solution for 4 hours at 0oC, then incubated in the Oslo chamber for one hour for recovery, then tested for K/Na ratio in the flame spectrometer.
Control slices showed an average ratio of about 2.83, with standard deviation of about 0.28 in the first series. After being in the CI washout solution as noted, the ratio in the first series averaged 2.36, S.D. 0.15 -- viability slightly less than controls. In the solution with HES the average ratio was 2.55, S.D. 0.22. There was no statistically significant difference between the solution with mannitol and the solution with HES. A repeat series of each yielded essentially identical results.
Comparing the new CI vehicle solution with m-RPS-2 was done similarly, with nearly identical results: no significant difference.
