The Brain Preservation Foundation’s Small Mammalian Brain Prize won

The Brain Preser­va­tion Foun­da­tion’s Small Mam­malian Brain Prize has been won with fan­tas­tic preser­va­tion of a whole rab­bit brain us­ing a new fix­a­tive+slow-vit­rifi­ca­tion pro­cess.

  • BPF an­nounce­ment (21CM’s an­nounce­ment)

  • evaluation

  • The pro­cess was pub­lished as “Alde­hyde-sta­bi­lized cry­op­reser­va­tion”, McIn­tyre & Fahy 2015 (mir­ror)

    We de­scribe here a new cry­obiolog­i­cal and neu­ro­biolog­i­cal tech­nique, alde­hyde-sta­bi­lized cry­op­reser­va­tion (ASC), which demon­strates the rele­vance and util­ity of ad­vanced cry­op­reser­va­tion sci­ence for the neu­ro­biolog­i­cal re­search com­mu­nity. ASC is a new brain-bank­ing tech­nique de­signed to fa­cil­i­tate neu­roanatomic re­search such as con­nec­tomics re­search, and has the unique abil­ity to com­bine sta­ble long term ice-free sam­ple stor­age with ex­cel­lent anatom­i­cal re­s­olu­tion. To demon­strate the fea­si­bil­ity of ASC, we per­fuse-fixed rab­bit and pig brains with a glu­taralde­hyde-based fix­a­tive, then slowly per­fused in­creas­ing con­cen­tra­tions of ethylene gly­col over sev­eral hours in a man­ner similar to tech­niques used for whole or­gan cry­op­reser­va­tion. Once 65% w/​v ethylene gly­col was reached, we vit­rified brains at −135 °C for in­definite long-term stor­age. Vitrified brains were re­warmed and the cry­opro­tec­tant re­moved ei­ther by per­fu­sion or grad­ual diffu­sion from brain slices. We eval­u­ated ASC-pro­cessed brains by elec­tron microscopy of mul­ti­ple re­gions across the whole brain and by Fo­cused Ion Beam Milling and Scan­ning Elec­tron Microscopy (FIB-SEM) imag­ing of se­lected brain vol­umes. Preser­va­tion was uniformly ex­cel­lent: pro­cesses were eas­ily trace­able and synapses were crisp in both species. Alde­hyde-sta­bi­lized cry­op­reser­va­tion has many ad­van­tages over other brain-bank­ing tech­niques: chem­i­cals are de­liv­ered via per­fu­sion, which en­ables easy scal­ing to brains of any size; vit­rifi­ca­tion en­sures that the ul­tra­struc­ture of the brain will not de­grade even over very long stor­age times; and the cry­opro­tec­tant can be re­moved, yield­ing a per­fus­able alde­hyde-pre­served brain which is suit­able for a wide va­ri­ety of brain as­says…We have shown that both rab­bit brains (10 g) and pig brains (80 g) can be pre­served equally well. We do not an­ti­ci­pate that there will be sig­nifi­cant bar­ri­ers to pre­serv­ing even larger brains such as bov­ine, ca­nine, or pri­mate brains us­ing ASC.

    (They had prob­lems with 2 pigs and got 1 pig brain suc­cess­fully cry­op­re­served but it wasn’t part of the en­try. I’m not sure why: is that be­cause the Large Mam­malian Brain Prize is not yet set up?)
  • pre­vi­ous dis­cus­sion: Mikula’s plas­ti­na­tion came close but ul­ti­mately didn’t seem to pre­serve the whole brain when ap­plied.

  • com­men­tary: Al­cor, Robin Han­son, John Smart, Ev­i­dence-Based Cry­on­ics, Vice, Pop Sci

  • dona­tion link

To sum­ma­rize it, you might say that this is a hy­brid of cur­rent plas­ti­na­tion and vit­rifi­ca­tion meth­ods, where in­stead of al­low­ing slow plas­ti­na­tion (with un­known de­cay & loss) or forc­ing fast cool­ing (with un­known dam­age and loss), a staged ap­proach is tak­ing: a fix­a­tive is in­jected into the brain first to im­me­di­ately lock down all pro­teins and stop all de­cay/​change, and then it is leisurely cooled down to be vit­rified.

This is ex­cit­ing progress be­cause the new method may wind up pre­serv­ing bet­ter than ei­ther of the par­ent meth­ods, but also be­cause it gives much greater visi­bil­ity into the end-re­sults: the alde­hyde-vit­rified brains can be eas­ily scanned with elec­tron micro­scopes and the re­sults seen in high de­tail, show­ing fan­tas­tic preser­va­tion of struc­ture, un­like reg­u­lar vit­rifi­ca­tion where the scans leave opaque how good the preser­va­tion was. This opac­ity is one rea­son that as Mike Dar­win has pointed out at length on his blog and jkauf­man has also noted that we can­not be con­fi­dent in how well ALCOR or CI’s vit­rifi­ca­tion works—be­cause if it didn’t, we have lit­tle way of know­ing.

EDIT: BPF’s founder Ken Hay­worth (Red­dit ac­count) has posted a piece, ar­gu­ing that ALCOR & CI can­not be trusted to do pro­ce­dures well and that fu­ture work should be done via rigor­ous clini­cal tri­als and only then rol­led out. “Opinion: The prize win is a vin­di­ca­tion of the idea of cry­on­ics, not of un­ac­countable cry­on­ics ser­vice or­ga­ni­za­tions”

…“Should cry­on­ics ser­vice or­ga­ni­za­tions im­me­di­ately start offer­ing this new ASC pro­ce­dure to their ‘pa­tients’?” My per­sonal an­swer (speak­ing for my­self, not on be­half of the BPF) has been a stead­fast NO. It should be re­mem­bered that these same cry­on­ics ser­vice or­ga­ni­za­tions have been offer­ing a differ­ent pro­ce­dure for years. A pro­ce­dure that was not able to demon­strate, to even my min­i­mal ex­pec­ta­tions, preser­va­tion of the brain’s neu­ral cir­cuitry. This re­sult, I must say, sur­prised and dis­ap­pointed me per­son­ally, lead­ing me to give up my mem­ber­ship in one such or­ga­ni­za­tion and to be­come ex­tremely skep­ti­cal of all since. Again, I stress, cur­rent cry­on­ics pro­ce­dures were NOT able to meet our challenge EVEN UNDER IDEAL LABORATORY CONDITIONS de­spite be­ing offered to pay­ing cus­tomers for years[1]. Should we re­ally ex­pect that these same or­ga­ni­za­tions can now be trusted to fur­ther de­velop and prop­erly im­ple­ment such a new, in­de­pen­dently-in­vented tech­nique for use un­der non-ideal con­di­tions?

Let’s step back for a mo­ment. A sin­gle, in­de­pen­dently-re­searched, sci­en­tific pub­li­ca­tion has come out that demon­strates a method of struc­tural brain preser­va­tion (ASC) com­pat­i­ble with long-term cryo­genic stor­age in an­i­mal mod­els (rab­bit and pig) un­der ideal lab­o­ra­tory con­di­tions (i.e. a healthy liv­ing an­i­mal im­me­di­ately be­ing per­fused with fix­a­tive). Should this one pa­per in­stantly open the floodgates to hu­man ap­pli­ca­tion? Un­der untested real-world con­di­tions where the ‘pa­tient’ is ei­ther ter­mi­nally ill or already de­clared legally dead? Should it be performed by un­li­censed per­sons, in un­ac­countable or­ga­ni­za­tions, op­er­at­ing out­side of the tra­di­tional med­i­cal es­tab­lish­ment with its checks and bal­ances de­signed to en­sure high stan­dards of qual­ity and ethics? To me, the clear an­swer is NO. If this was a new drug for can­cer ther­apy, or a new type of heart surgery, many ad­di­tional steps would be ex­pected be­fore even clini­cal tri­als could start. Why should our ex­pec­ta­tions be any lower for this?

The fact that the ASC pro­ce­dure has won the brain preser­va­tion prize should rightly be seen as a vin­di­ca­tion of the cen­tral idea of cry­on­ics –the brain’s del­i­cate cir­cuitry un­der­ly­ing mem­ory and per­son­al­ity CAN in fact be pre­served in­definitely, po­ten­tially serv­ing as a life­sav­ing bridge to fu­ture re­vival tech­nolo­gies. But, this mile­stone should cer­tainly not be in­ter­preted as a vin­di­ca­tion of the very differ­ent cry­on­ics pro­ce­dures that are prac­ticed on hu­man pa­tients to­day. And it should not be seen as a man­date for more of the same but with an alde­hyde sta­bi­liza­tion step ca­su­ally tacked on. …