The Institute for Neural Cryobiology
 
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Cryopreservation of Hippocampal Slices

Abstract

This project seeks to determine optimal methods for preserving brain slices, in particular hippocampal slices, at ultralow temperatures. Slices preserved by these methods may have potential future applications for research into schizophrenia, Alzheimer's disease, Parkinson's disease, and ischemic brain damage. These methods could also 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 (which happens will depend on the concentration of the cryoprotectant formula used) 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, speed of spread, area, and duration of depolarization. The number of discreet visual depolarization or physical defects (holes) per field will also be counted.

Host Institution and Financing

The project has recently been approved for joint financing by INC and Harbor-UCLA Research and Education Institute (REI) which will host the project experimental work. Under the terms of a contract with INC, REI will provide approximately $75,000 for salaries and supplies, and INC will purchase and supply equipment costing approximately $77,000 which will be required by the project. For details see the Budget, and Funding our Work

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 are available on request and by signing a non-disclosure agreement.

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

Principal Investigator, Dr. Robert J. Morin

The Principal Investigator of the Project will be Dr. Robert J. Morin, Research Professor of Pathology at REI and Chairman of the Department of Pathology at Harbor-UCLA Medical Center. Dr. Morin will coordinate the proposed research by supervising and advising the project experimentalist in conjunction with Dr. Fahy. Dr. Morin has extensive expertise in both project management and in pathological processes germane to the project.

Project Experimentalist

A scientist or technician familiar with brain slice preparations will be hired to conduct the experimental aspects of the project. A secondary consideration will be experience in cryobiology.

Consulting Investigator, Dr. Gregory M. Fahy

Dr. Fahy was instrumental in the protocol design, and will be Consulting 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 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 LRT, 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:                    Project experimentalist, full-time
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 Harbor-UCLA Research and Education Institute
---------
$74,689
Equipment:   Axon Imaging Workbench 2.0, Sigma Plot, SigmaStat
4,500
      Image recording system: MotionScope plus frame grabber PCI 2000
25,500
      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, for positioning nanodrop, Fine Science MM3, support
1,085
      Cryo-Med controlled rate freezer
15,000
      Pentium II PC, 266 MHz , 64 MB RAM, 4GB Disk, Zip Drive, CD 
3,000
Total Cost of Equipment purchased & supplied by the Institute for Neural Cryobiology
---------
$76,925
Total Project Cost $151,614
 
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