X-Message-Number: 9388
Date: Wed, 01 Apr 1998 22:27:07 -0800
From: Paul Wakfer <>
Subject: Cryopreservation of Hippocampal Slices
The Institute for Neural Cryobiology (INC) is a California Nonprofit
Public Benefit Corporation formed for the purpose of promoting
scientific research specifically related to the cryobiology of nerve
tissues. This includes both conducting such research and aiding the
publication of the research of others as well as our own. A major
purpose of the Institute is to research and develop methods for the
damage-free preservation of neural tissue to allow long-term stable
storage at cryogenic temperatures (below -80'C) for neuroscience and
medical purposes.
Our website at http://neurocryo.org is currently under construction, but
will soon contain many details and addition to the information which is
presented here.
INC is pleased to present for your consideration its first research
project.
Cryopreservation of Hippocampal Slices.
Abstract
This project seeks to determine optimal methods for preserving brain
slices (hippocampal slices) at ultralow temperatures. Slices preserved
by these methods may have potential future applications for research
into schizophrenia, Alzheimer's disease, Parkinson's, and ischemic brain
damage. These methods could greatly reduce the numbers of experimental
animals required for this type of research. Preservation of such slices
in banks at low temperature and the distribution of model slices would
also allow the study of perishable human material by pathology students
and scientists to proceed with greater ease.
Slices will be prepared and maintained using standard procedures. They
will be exposed to four different cryoprotectant formulae
(cryoprotectants are chemicals that reduce or prevent freezing injury)
according to an optimized two-step method, then either frozen or
vitrified using a variety of cooling and warming rates, temperature
minima, and storage times. After cryopreservation, the slices will be
rewarmed, washed to remove the cryoprotectants, and tested for normal
electrical activity and fine structure. Testing will involve chemical
stimulation (glutamate and potassium) to elicit action potentials, and
detection of the pattern of elicited electrical activity by means of
voltage-responsive dyes and high speed videomicrography. Videotape
frames will be subjected to semi-automated image analysis for
quantitation of the magnitude of depolarization, the speed of spread of
depolarization, the area of depolarization, and the duration of
depolarization. The number of discreet visual depolarization or physical
defects (holes) per field will also be counted.
Project Financing
The project is to be financed jointly with a major university medical
center Research Institute (RI) which will pay approximately $75,000 for
salaries and supplies. The Institute for Neural Cryobiology (INC) will
purchase and supply equipment costing approximately $75,000 which will
be required by the project. The advantage to INC in purchasing and
supplying this equipment is two-fold. First, INC will retain ownership
of the equipment and, thus, will be able to use it for ongoing research
wherever that might take place. Second, INC will not have to pay the
standard 27.5% overhead which RI charges on any money contributed to it
before that money is applied to project funding. Currently (April 1,
1998), INC has $30,000 of donated funds available to finance its portion
of the project. INC is seeking additional donations to increase this
total to meet its $75,000 commitment. Since INC is a California
Nonprofit Public Benefit Corporation, receipts for donations to INC by
US taxpayers may be used to reduce taxable income.
Details of Research Plan
Overall objective or aims
The goal of the proposed research is to understand the cryobiology of
the mammalian hippocampal slice and to develop effective means for its
cryopreservation.
Results of this research may lead to development of methods for
preservation of uniform brain slices from both normal and pathological
specimens with preservation of morphology, biochemical viability,
neurophysiological function, and pharmacological responsiveness, which
could be made commercially available as experimental models for various
research protocols. This could greatly reduce the numbers of animals
required for experimentation and thereby reduce costs. Specialized
cryopreserved brain slices could also have potential future medical
applications.
Background
There are nearly no reports on the recovery of integrated neural
functions such as synaptic transmission and action potential propagation
after the cryopreservation and rewarming of central nervous system (CNS)
neural pathways. One exception is Pascoe's work showing successful
preservation of synaptic function after freezing and thawing the
superior cervical ganglion, but this may or may not reflect the
responses of CNS structures important for the understanding of brain
function and pathology.
The hippocampus is an ideal model for such investigations. The
hippocampus is specifically affected in Alzheimer's disease, amnesias,
schizophrenia, epilepsy, and post-ischemic injury associated with
cardiac arrest, has been well-studied both anatomically and
functionally, and is easy to identify and surgically isolate.
Hippocampal slices are an established neurobiological preparation, and
can be maintained in culture for days to weeks. Because approximately 8
slices can be made from the dorsal hippocampus of a single rat (4 slices
from each hemisphere), use of the slice model allows results to be
obtained relatively rapidly.
Glycerol, dimethyl sulfoxide (DMSO), DMSO plus formamide, and, to a
lesser extent, a mixture of 2,3-butanediol and 1,2-propanediol (BP) have
cryoprotective effects for isolated CNS neurons and synapses. Intact
rat brains, however, take up cryoprotective agents (CPAs,
cryoprotectants) poorly. A brain tissue slice model will eliminate any
contribution of the blood brain barrier to this failure of CPA uptake.
No useful information is available on the ideal cryoprotectant
selection, concentration, introduction temperature, rate of
administration, elution rate, elution osmolyte, or elution temperature
for organized neural systems, nor is there information on the ideal
phase change duration, post-isotherm cooling rate, plunge temperature,
storage temperature, warming rate, or warming method, nor information on
prospects for neural system vitrification.
Vitrification, originated by Fahy, is another approach to
cryopreservation which allows ice formation and dissolution to be
entirely avoided. After 17 years of development, enough has been learned
to make the vitrification of hippocampal slices an attractive
possibility.
Rationale behind approach
The possibility of difficulty in attaining good CPA permeation as well
as the likely sensitivity of the hippocampal slice to disruption by ice
grains suggest the need for optimization of CPA administration. For this
reason, the approach of Rall and Fahy will be followed. This method is
described below. Only after cryoprotection is optimized can the best
results be obtained by examining the standard cryobiological variables
involved in freezing and thawing, or in vitrification.
The assay method used in this study should survey the hippocampal slice
as a whole, not merely the responses of single cells or particular
points in space, because injury may be distributed stochastically. The
method selected is therefore visualization of global neural activity
using light microscopy and the voltage-stimulated change in the
fluorescence of the dye, RH-414. RH-414 has been used successfully for
imaging hippocampal preparations and emits more light than is absorbed
by RH-155 which has been used successfully to image waves of neural
activity in the hippocampus with sub-milisecond time resolution and good
spatial resolution. Commercially available equipment will allow even
greater spatial resolution to be obtained in the proposed studies.
Specific aims and detailed methods of procedure
Note: These highly technical details are intellectual property which may
be patentable and cannot be presented in full at this time.
Data analysis
Quantitative analysis of the data will require normalization to
compensate for varying slice thicknesses and other ponderable and
imponderable differences that have been reported from slice to slice. At
a minimum, end points such as propagation rate, depolarized area, etc.
will be subjected to testing of the difference between means using
standard methods (t-test or, if the assumptions fail, the Mann-Whitney
rank sum test). SigmaStat, a statistical software package, will be used
to calculate appropriate group sizes for significance should the
allotted 4 slices/group norm be insufficient.
Data analysis will constitute a major portion of the labor associated
with this project. Given 4 experimental working days per week, the work
described would require about 30 weeks to complete. However, each
experiment will generate about 600 usable frames of videotape that will
require analysis for each of 4 variables (magnitude, propagation rate,
and area of depolarization as well as the number of clear physical
defects present in the field of view) for each of three hippocampal
subregions, before and after the induction of LTP, for a total of 24 end
points per experiment. This analysis and graphical representation of the
data plus the examination of the electron micrographs will occupy the
balance of the funding period for this project. The analysis will be
semi-automated using Axon Imaging Workbench 2.0, a commercial program
specifically designed for this purpose.
Expertise and roles of investigators in the specific area of study
Dr. X
Dr. X who wishes to remain publicly anonymous at this time, is Professor
and Chairman of the Department of Pathology at the major medical center
where RI is located. Dr. X will be the co-Principal Investigator of the
study. In this role, he will coordinate the proposed research by
supervising and advising Dr. Pichugin in conjunction with Dr. Fahy. He
has extensive expertise in both project management and in pathological
processes germane to the project.
Dr. Yuri Pichugin
Dr. Pichugin will conduct the experimental aspects of the project. Dr.
Pichugin has 20 years of laboratory experience in the field of tissue
cryopreservation and cryoprotection, and has worked at the largest
cryobiological institute in the world, in Kharkov, during most of his
career. In recent years, he has specifically studied the
cryopreservation of central nervous system tissue. Dr. Pichugin has
published extensively on mechanisms of cryoprotection and cryoprotectant
toxicity.
Dr. Gregory M. Fahy
Dr. Fahy was instrumental in the protocol design, and will be
co-Principal Investigator to the project. Dr. Fahy has studied the
cryopreservation of mammalian organs since 1972 and has an extensive
background of discovery in cryobiology. He was the first to show that
cryoprotectants exert toxicity in the frozen state, and he was the
originator of a practical approach to cryopreserving cells, tissues, and
organs without freezing (vitrification) which has found extensive use
for a myriad of cells and tissues ranging from plant cells to animal
embryos to human pancreatic islets. He published the first successful
cryopreservation of viable peripheral nerves and the first paper on
delivering cryoprotectants to nervous tissue by continuous gradient
perfusion. He has invented numerous devices required for cryoprotectant
perfusion and is the holder of several patents on these devices. He was
also instrumental in the writing of a recent NIST ATP grant on tissue
cryopreservation for $2.5 million which was awarded to Life
Resuscitation Technologies, Inc., of which he was Chief Scientist. Since
1994, he was also the Chief Scientist of Organ, Inc., for which he
developed novel technologies for the control of ice crystallization
(four patents pending). Dr. Fahy has observed the hippocampal dissection
and slicing technique at the Uniformed Services University for the
Health Sciences in the laboratory of Dr. John Sarvey, who will further
advise the project on this technique as and if necessary.
Budget
Salary: Dr. Yuri Pichugin, Research Associate, 40 hours per
week 40,000
Cost of Fringe
Benefits 12,800
Supplies:
Videotapes 150
Anaesthetics,
cryoprotectants 3,500
Perfusates and artificial cerebrospinal
fluid 8,000
Liquid Nitrogen and
carbogen 400
Suture, cannula tubing, rat disposal
bags 250
Glassware, plasticware (fabrication of 8 Scot
chambers) 2,000
RH-414 fluorescent
dye 250
Animal Purchases: 130 rats @
$10.50/rat 1,365
Animal Services: 14 days/rats @
$0.70/day 1,274
Publication Cost: one publication with color figures, page and reprint
charges 3,200
Travel: Two trips for training plus one
presentation 1,500
-------
Total Cost to be paid by
RI $72,050
Equipment: Axon Imaging Workbench 2.0, Sigma Plot,
SigmaStat 4,500
Image recording system: MotionScope plus frame grabber
PCI 2000 27,000
Zeiss microscope with epifluorescent
capability 23,000
Brain slice chambers (2), Fine Science, with
support 2,840
Superfusion equipment, sterilizing filters plus roller
pump 2,000
Micromanipulator, positioning nanodrop, Fine Science
MM3 1,085
Cryo-Med controlled rate
freezer 15,000
Pentium II PC, 300 MHz, 64 MB RAM, 4GB Disk, Zip Drive,
CD 3,000
-------
Total Cost of Equipment purchased and supplied by
INC $73,925
=======Total Project
Cost
$145,975
For more information contact the undersigned.
Paul Wakfer
Voice/Fax: 909-481-9620 Page: 800-805-2870
The Institute for Neural Cryobiology - http://neurocryo.org
Perfected cryopreservation of Central Nervous System tissue
for neuroscience research and medical repair of brain diseases
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