Part 2: The experimental study of ischemia of cerebral tissues.

By Yuri Pichugin, PhD

Another of my hypotheses was that higher survival of the hippocampal slices may be as a result of that they completely had no warm ischemia in comparison with the rat brains. To verify this hypothesis, I performed the experiments with the cerebral slices which had 2, 6, and 10 minutes of warm ischemia plus 6 hour cold ischemia. The results of these experiments are presented in Fig. 4. Even short time warm (30°C) ischemia of cerebral slices can rather significantly decrease their survival. However, ischemic exposure for the hippocampal slices was acuter than it was for the whole brains because the very thin slices, which were placed in physiological solution with lack of oxygen and glucose at 30°C, were very quickly washed out from oxygen and glucose.

Fig. 4 Combination of 0-10 minute warm ischemia with 6 hour cold ischemia for the rat cerebral slices.

Thus, higher survival of the hippocampal slices (Table 2) was a result of that they completely had no warm ischemic exposure in comparison with the rat brains.

5. Testing some compounds on rat brains to decrease negative effects of long time cold ischemia.

The June 2005 issue of Scientific American reported about "Suspended Animation" of mice by hydrogen sulfide gas (H₂S). (Also, see Hydrogen Sulfide for Cryonics? By Ben Best (The Immortalist, v. 37, No.7-8, p. 14, 2005). Some of cryonicists asked the question, Could hydrogen sulfide be an excellent additive to organ preservation solutions to prevent cold ischemia?

I tested various H₂S concentrations (200 mg/L to 2 mg/L) using rat brains that were perfused with cold aCSF solutions contained the H₂S concentrations and kept at 2-4°C for 24 hours. H₂S showed no positive effect in comparison with the control (43±2%, see Fig. 5).

Fig. 5 Effect of hydrogen sulfide on the rat brains perfused with the various amount of H₂S and stored at 2-4° C for 24 hours. (The squares without error bars indicate only single experiments, n=1). H₂S concentrations from 100 mg/L or higher began to manifest toxicity for the rat cerebral tissues.

The CI research project that the CI President, Ben Best proposed me a year ago concerned testing organ preservation solutions and improving them for 24-48 hour cold storage of the brain. I said to him that the research theme was a very difficult one. Many world-class research labs work on the study and improvement of organ preservation solutions and it is a multi-million dollar business. It is necessary to vary types and concentrations of components of organ preservation solutions such as salts (cations and anions), energy sources, colloids, buffers and some potential useful additives such as ROS scavengers (antioxidants), 2^nd messengers, NO donors, metabolism inhibitors and so on. This theme can keep me busy for 20 years and more! Of course, the purpose was to improve cold preservation solutions for 24 hour storage of the brain to obtain not 100% survival but if only 70-80% survival instead of 43% one. But this was impossible to reach. I tested glycine (Fig. 6), glycine plus H₂S (Fig. 6), N-acetylcysteine (Fig. 7), and deteroxamine (Fig. 8).

Fig. 6 Testing H₂S, glycine, and glycine plus H₂S for cold storage of the rat brains. 1 – the untreated brains; 2 – the brains perfused with aCSF; 3 – the brains perfused with aCSF plus 2 mg/L H₂S; 4 – the brains perfused with aCSF plus 10 mM/L glycine; 3 – the brains perfused with aCSF plus 10 mM/L glycine and 2 mg/L H₂S;

None of the compounds showed a positive effect in comparison with the control, untreated rat brains. The compounds had a little positive effect for 6 hour cold storage in comparison with aCSF perfusion as a second control. For 3 hours, the combination of glycine and H₂S preserved the brains by 20% better than the aCSF control (the column #2).

Fig. 7 Effect of N-acetylcysteine (XXXX) on the rat brains perfused with the dose of 400 mg/kg N-acetylcysteine and stored at 22oC for 1-2 hours in comparison with the aCSF control (/////).

Fig. 8 Effect of deteroxamine (XXXX) on the rat brains perfused with 0.625 mM/L deteroxamine and stored at 2-4oC for 3 or 6 hours in comparison with the aCSF control (/////). Deteroxamine had a little positive effect for 3 hour cold storage in comparison with aCSF perfusion (the aCSF control). Ben Best also proposed to test cyanide, rotenone, acetyl-dl-carnitine (Alcar), lipoic acid together with Alcar, dextromethorphan, and PEG 20,000. We can find tens or even hundreds compounds in scientific literature that may be probably useful for improving cold storage solutions.

Fig. 8