This presentation outlines the major techniques, approaches and models employed in the study of neuroregeneration especially of the central nervous system. The most promising avenues of research involve neurotrophic factors isolated from mildly damaged nerve tissue and the use of embryonic tissue. Other therapeutic medicines have also proven helpful in reducing damage by anti-inflammatory effects or causing an insulative effect to reduce the effects of demyelination. Cloning of plasmid vectors of cDNA for neurotrophins is important in collecting medicinal dosages of these neurotrophic factors. Embryonic cloning of adult human tissue whether gremlin or somatic may also increase neuroregeneration over that of unrelated fetal tissue transplantation. Such adult human cloning of embryonic tissue could also be used to create a body double for organ transplants. Even brain transplantation would be possible if neuroregeneration of severed spinal columns are successful in humans, as they have been in laboratory rats.

Techniques of Neuroregeneration:

(and possible clinical medical application)

I Molecular Neuroscience

A Isolate mRNA by Northern blot and proteins by Western blot that are expressed in damaged nerve tissue and not control nerve tissue of the same species without the same injury

Variables that appear critical:

1. Cell cycle stage
2. Developmental stage
3. Dosage of neurotrophins and the relative amount, timing, order of injection of the neurotrophins if more than one is administered
4. Species used

C Reverse transcribe RNA hybridized to cDNA

D Sequence the cDNA by dideoxy chain termination for both intra- and interspecies comparison to possible homologies in GenBank and clues as to receptor binding affinities and mechanism of action.

E Sequence the protein by use of Edmund's reagent for structural and functional clues as to its mechanism

F Transfect the cDNA into a plasmid vector to manufacture a relevant dosage to determine its neuroregenerative properties under specific controlled conditions.

II Immunoneuroscience Technique

A Isolate a suspected inhibitory factor

B Generate an antibody to it by injecting it into another animal (often rabbits)

C Inject the antibody and also at least one neurotrophin e.g. NT3.

D If neuroregeneration occurs with antibody and not without (or a significant increase in regenerative rate occurs), the protein factor is likely an inhibitor factor.

E Reason for inhibitory factors

1. When the various organs of the body plan are finished, the chemical messengers driving this differentiation must be shut off.
2. Neurons appear to be the source of differentiation beyond the stage of gastrulation and the cause of organogenesis.
3. Temporarily re-starting the embryonic stages of nerve proliferation appear to occur in invertebrates more easily than vertebrates. The feedback mechanism involved in this may have been damaged or mutated during evolution.
4. The regeneration of brain cells in particular can not happen at a high rate or the synaptic network that provides a stable thinking pattern would be disturbed. If the regeneration included glial cells, memories would be lost.

III Therapeutic techniques

A An anti-inflammatory drug called methyprednisolone reduces the extent of damage after injury (optimal results only occur if administered within 8 hours of injury)

B The drug 4-amino pyridine (4AP) acts as a chemical sheath surrounding the exposed surfaces of demyelinated axons from injury thus allowing better conductance of neuronal electrical signals.

C X-ray radiation also has been shown to cause the activation of neuroregeneration by some unknown mechanism.
 
 

IV Embryonic tissue transplantation

A Only 5-10 % of the spinal chord is necessary for motor functioning in humans and other test animals e.g. rats

B The regenerating of crushed axons that are beyond repair can be replaced by embryonic tissue transplantation

  V An integrated strategy, re-configuring the embryonic stage of neurulation:

It would appear to me that embryonic tissue, neurotrophins and a neural bridge from related neural cells elsewhere in the patients body combined would make a viable protocol to potentially reconnect at least 5 to 10% of a severed spinal column thus allowing for recovery of motor function.

A The "neural bridge" spans the gap as was successfully done at the Karolinska Institute in laboratory rats. The bridge behaves, most likely, as a necessary substratum along which to migrate. The neural bridge has the extracellular matrix receptors needed to guide the axons along this path as described in neurulation stage of embryology.

B The neurotrophins provided supply the messages that the embryonic neural tissue would have supplied during neurulation.

C The embryonic tissues also respond to the neurotrophins and extracellular matrix due to their early developmental stage as undifferentiated cells and provide replacement of permanently damaged axons (called "collateral sprouting or reactive reinnervation). These embryonic cells may then also contribute to the secretion of appropriate neurotrophins as they are so signaled by the externally supplied factors.

D The embryonic cloning of one's own adult, diploid germ line tissue would likely improve the rate and success of this "neural bridge" design for spinal chord regeneration. This can be accomplished by "diploid nuclear replacement of mature human ova".

E The active genes for the production of various neurotrophins can be spliced into neurons that are then injected into the damaged site.
 
 

VI Adult Human Cloning of One's Own Embryonic Tissue: The Technique Used by Shettles and Wilmut (with minor variations in the nuclear donors)

A Nuclear donors

  Shettles uses diploid germ line precursor cells from an adult man called spermatogonia (oogonia for women) as the nuclear donor

whereas
1. Wilmut uses somatic cells from the udder of a lamb and starves it with a 0.5% instead of 10% serum in the culture medium to arrest its cell cycle in the G0 phase. Halting the growth phase provided enough time for the cytoplasmic determinants of the recipient egg in metaphase II to reprogram the donor nucleus. Unfertilized eggs have had a higher cloning success rate than enucleated zygotes. (xii)

C Shettles cultured the human oocytes in its own follicular fluid whereas Wilmut utilized both ligated oviducts and a chemically defined serum medium to culture his eggs.

D Shettles terminated his experiment at the morulla stage noting normal morphology. Shettles noted that at the morula stage the fetus is ready for implantation. Wilmut transferred most of his fetal cells to recipient ewes at either the morula or blastocyst stage. When surplus fetuses were available to Wilmut, selection was based on best morphology.

E This nuclear transfer technique could be used to make a body double for each human that wanted and could finance one.

F Surplus embryos to store for later neuroregenerative therapy (e.g. of a severed spinal column from body transplantation) could be made using the Hall-Stillman technique.

 VII Adult Human Cloning to Completion Allows for Body Transplantation without Quadriplegia if Neuroregeneration of a Severed Spinal Column is achieved

A Preliminary considerations

1. Surgical removal of the telencephalon at 6 weeks gestation would be needed for vegetative reproduction of a body double instead of a twin.
2. Supernutrient feeding and growth hormones would expedite its development rate after anencephalic birth.

1. Blood sinuses must be cauterized to prevent undesired leakage.
2. This interposition technique allows for continuous blood flow to prevent ischemia
3. The only problem noted besides the quadriplegia was death due to immunological rejection. Such rejection could not occur with identical body parts and therefore identical MHC that becomes possible with the Shettles-Wilmut nuclear replacement technique of cloning mammals.

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Neuroregeneration:

Baffour, Richard et al. "Synergestic effect of basic fibroblast growth factor and methylprednisolone on neurological function after experimental spinal cord injury" Journal of Neurosurgery Volume 83, pages 105-110.

Bregman, Barbara S. et al. "Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors" Nature Volume 378, pages 498-501.

Cheng, Henrich et al. "Spinal Chord Repair in Rats: Partial Restoration of Hind Limb Function" Science Volume 273, pages 510-513.

Donnerer, J. "Improved neurochemical recovery of 6-hyroxydopamine-lesioned postganglionic sympathetic neurons by nerve growth factor in the adult rat" Neuroscience Letters 1996, Volume 221, pages 33-36.

Donnerer, J et al "Complete recovery by nerve growth factor of neuropeptide content and function in capsaicin-impaired sensory neurons" Brain Research 1996, Volume 741, pages 103-108.

Gold, Bruce G. et al. "The Immunosuppressant FK506 Increases the Rate of Axonal Regeneration in Rat Sciatic Nerve" Journal of Neuroscience November, 1995 Volume 15, Number 11, pages 7509-7516.

Hellweg et al. "Moderate lesion of the rat cholinergic septohippocampal pathway increases hippocampal nerve growth factor synthesis: evidence for long-term compensatory changes?" Molecular Brain Research 1997, Volume 45, pages 177-181.

Shahar, Abraham et al. "Tissue Culture Models in the Study of Neuroregeneration and Neuroregenerative Processes" Journal of Neuropathology and Experimental Neurology 1995 Supplement pages 20S-21S.

Windle. W.F. et al. "Risidual function after subtotal spinal cordtransection in adult cats" Neurology 1958 Volume 8, pages 518-521.

Young, Wise "Spinal Chord Regeneration" Science Volume 273, page 451.

 Adult Human and Mammalian Cloning:

Shettles, Landrum Beatrice, MD, Ph.D., F.A.C.S., F.A.C.O.G.., F.R.S.H.. "Diploid nuclear replacement in mature human ova with cleavage" American Journal of Obstetrics and Gynecology January 15, 1979,

Steward, Colin (review) "Nuclear Transplantation, An udder way of making lambs" Nature 27 February 1997, Volume 385, pages 769-770.

Wilmut, Ian et al. "Viable offspring derived from fetal and adult mammalian cells" Nature 27 February 1997, Volume 385, pages 810-813.

Brain Transplantation:

White, Robert, M.D., Ph.D. et al. "The isolation and transplantation of the brain. An historical perspective emphasizing the surgical solutions to the design of these classical models" Neurological Research Volume 18, pages 194-203.

White, Robert, M.D., Ph.D. "Brain transplantation: Prolonged survival of brain after carotid-jugular interposition" Science Volume 150, pages 779-881.

White, Robert, M.D., Ph.D. "Cephalic exchange transplantation in the monkey" Surgery July, 1971, Volume 70, Number 1, Pages 135-139.

White, Robert, M.D., Ph.D. "Experimental Transplantation of the Brain" chapter 44 in Human Transplantation (no further information found)

White, Robert, M.D., Ph.D. "Primate cephalic transplantation: neurogenic separation and vascular association" Transplantation Proceedings March 1971, Volume 3, pages 602-604.

A Modest Proposal?

Segall, Paul, Ph.D. and Carol Kahn "Cloning: Born Again" chapter 4 in Living Longer, Growing Younger, Remarkable Breakthroughs in Life Extension 1989, Random House, New York

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