ACQUISITION, PREPARATION, and PRESERVATION of NEURAL TISSUE.

  PRE-OPERATIVE PREPARATION of PATIENT (Imaging the site).

SURGICAL PROCEDURE

PERI-OPERATIVE MANAGEMENT.

EVALUATION.

MODIFICATION OF PROCEDURAL PARAMETERS.

FACTORS IN GRAFT SURVIVAL.

OBSERVED SIDE EFFECTS.

CLINICAL RESULTS of PORCINE NEURAL XENOTRANSPLANTATION.

FUTURE GOALS and MODIFICATIONS TOWARD OPTIMIZATION.

REFERENCES.
 

ACQUISITION, PREPARATION, and PRESERVATION of NEURAL TISSUE

(Freeman 1995, Dunnet 1992)
 

Acquisition

                                • The VM tissue is a good source of dopaminergic (DA) neurons, or nigrostriatal neurons, which have shown

                                   to have the ability to reestablish lost neural connections as in the recipient rat striatum. (Glapern 1996)

                                • Pig-derived embryonic tissue is used because: (Widner 1999)

• Sufficient cell number available
                             • Proper size of neurons

                           • Relatively inexpensive

                           • The dopaminergic system of the domestic pig is relatively known.

                           • The pig can be transgenically modified. (Ostergaard 1992)
 

  Preparation

                                • Immediately before transplantation, the tissue is further dissected into 3/4 mm³ pieces and chilled in a Hanks Balance

                                  Salt Solution (HBSS).

  Preservation

                                                • Survival for a few days.
 
                                                • Marked loss of DA neurons.

                                • Storage which Lowers Cell Metabolism

                                                • Cryopreservation (Freezing)
 
                                                                   • Leads to a decrease of 65-75% in neuronal survival of fresh control values.
 
                                                 • Hibernation

PRE-OPERATIVE PREPARATION of PATIENT (Imaging the site)

(Freeman 1995, Palfi 1997)
 

  Patients are placed in a standard magnetic resonance imaging (MRI)-compatible stereotactic frame using local
 
      anesthesia.

  The implantation site (using the post commissural putamen as an example) is visualized with MRI using a fast-spin

      echo sequence.

  Axial images are taken in 3 mm sections from below the putamen to above the caudate.

  Coronal images are also taken at 3 mm sections from 3 cm anterior to the coronal suture and then progressing caudally

       through the putamen.

  Implantation sites are determined by the "zero point" in the putamen which is defined as the half way point between

      its rostral  and caudal aspects on the lowest axial section.
 
   All other target sites are based on this single stereotactic measurement.

   NOTE: Success of the transplantation is determined in part by the accuracy in hitting the precise

       implantation site. It is difficult to obtain a reliable delineation of the three subregions of the putamen
 
       (pre-commissural, commissural, and post-commissural) using classical stereotactical mapping techniques. The

       technique developed  by  Palfi et al allows precise and reproducible identification of these three regions. This is

       beneficial by not only increasing the efficacy of the procedure, but also by minimizing the interference of the
 
       inter-individual variability in the interpretation of clinical results.

SURGICAL PROCEDURE

(Freeman 1995)
 

  In the operating room, the stereotactic grid array is aligned so that its axial plane is parallel to the axial plane of

      the MRI and so the longitudinal axis is parallel to the axis of the midline of the brain. (This allows for proper

      placement, coordinates and angle, of the stereotactic needle.)

  A burr hole into the skull is created in order to accommodate the site of entry of the transplant needle which is

      positioned so that the superficial needle tract remains entirely within the superior frontal gyrus.
 
 

  An optimal length and diameter of the transplant needle should be chosen.
 

  The dissected porcine tissue is aspirated into the stereotactic needle.
 

  The transplant needle is placed into the "zero point" on the grid array and directed to the "zero point" of the putamen.
 

  Each needle tract contains tissue from half the mesencephalon (one substania nigra).
 

  Each needle tract consists of four deposits in a volume of 4-5µL of HBSS and are implanted at 5 mm intervals.
 

  Injections should be made at a rate of 2µL / 30 seconds with an interval of 1 minute between deposits.
 

  Following the last deposit in each tract, 3 µL of HBSS is injected and the needle is left in place for 2 minutes in order

  Subsequent needle trajectories use the same burr hole and entry point by angling the grid array.
 

  A total of 6 to 8 needle tracts are made on each side (if performing a bilateral procedure) using 3 to 4 embryos per

  Surgery can be separated into two procedures.

PERI-OPERATIVE MANAGEMENT

(Freeman 1995)
 

Immunosuppression with Cyclosporin A should be initiated two weeks before the first transplantation procedure,

     should be a bit reduced at 2 weeks after the second procedure and discontinued after 6 months.

The use of anti-Parkinson medication should be tentatively halted a few days before surgery.   Antibiotic treatment with either piperacillin or vancomycin, ceftezidine, and flurconazole should be given by IV

     immediately after surgery and then continued for at least 5 days and as long as necessary.

  As a safety  measurement, as well as to test for breakdown of the Blood Brain Barrier , an MRI should be performed

      on the first post-operative day.

  After surgery, anti-Parkinson medication should be re-instituted. (Although this action is being re-evaluated due

      to the observed occurrence of adverse affects.)

 
 
 

EVALUATION

(Freeman 1995)
 

Positron Emission Tomography (PET) scans taken and analyzed in order to determine the fluorodopa (FD)

     uptake rate constant (K_i ). This is then compared to previous FD-PET scans taken in normal and in Parkinson’s

     patients who have not had the operation.

Core Assessment Program For Intracerebral Transplantation (CAPIT)

 • Unified Parkinson’s Disease Rating Scale (UPDRS)

 • The degree of dyskinesias

 • Hoehn-Yahr

  • Schwab-England
 

  Coordination, Dexterity, and Strength Tests

  Diurnal ("on-off" phenomena) (Wenning et al)

 

 

MODIFICATION OF PROCEDURAL PARAMETERS

(Freeman 1995)

Possible Implantation sites

                • Post Commissural Putamen (PCP) vs Caudate Nucleus

                                • Both autopsy and PET studies in Parkinson’s Disease demonstrate a greater dopamine depletion

                                  within the posterior putamen than in the anterior putamen caudate nucleus.

                                • Degeneration of the substania nigra preferentially occurs in regions that project to the posterior

                                  putamen.

                                • The PCP receives more input from the precentral motor fields

                                • Microstimulation studies within the PCP evoke discrete movements of contralateral body parts
 
                                  whereas the anterior putamen and caudate nucleus receive input primarily from the prefrontal
 
                                   cortex and frontal eye fields.

                                 • Nucleus Basalis of Mynert

                                 • Locus Ceruleous

                                 • Nigra Pars Compacta
 

  Bilateral vs Unilateral Implantation

                                    sides in some patients . This is postulated to be due to the continuous degeneration of the non-grafted side

                                    deleteriously affecting the operated side.
 

Alternative graft techniques (Borlongan 1998)

                   • Encapsulated cells

                   • Genetically engineered cells

 

 

(Dunnet 1992)

Immunological-based parameters

Location of implantation site (putamen vs caudate, unilateral vs bilateral)

Gestational age of donor tissue

Sterility

Method of transplantation (solid state vs cell suspension)

Presence of a lesion due to a previous operation (Pakzaban 1994)

Volume and amount of tissue implanted

Progression of the patient’s disease

 

 

(Peschanski 1994)

Psychological disorders (Obsessive compulsive disorder, auditory hallucinations, echo phenomena,

     depression, insomnia).

Frontal symptoms (slight euphoria).

Variance of dyskinesias.

Worsening of Parkinson’s symptoms.

Transplant has been observed to interact with L-Dopa therapy in a toxic effect on transplanted cells leading to a rapid

    decrease in L-Dopa intake after grafting.

Fatal of handicapping intracerebral hemorrhages and abscess. (Palfi 1998)

CLINICAL RESULTS of PORCINE NEURAL XENOTRANSPLANTATION

Deacon et al, 1997.

                • Method and Protocol:

                                • Implantation of approximately 600,000 dopaminergic pig neurons into one patient.
 
                                • Low doses of cyclosporine A were given.

                • Results:

                                • The patient did not exhibit any clinical benefits and died of a pulmonary embolism 7 months
 
                                  after surgery.
 
                                • Upon autopsy, only 628 grafted cells remained in the brain.

                                • It has been proposed that an immune reaction against the transplanted neural tissue was responsible

                                  for graft destruction.

Ellias et al, 1997.

                • Methods and Protocol:
 
                                • 12 patients with moderate to severe Parkinson’s Disease were treated one of whom was the same

                                    patient as in the previous study.

                                • Unilateral transplantation into 1 site in the caudate and 2 sites in the putamen.

                                • 6 patients received cyclosporine immunosuppression and 6 patients received cells which were

                                  treated with an F(ab’)₂ antibody fragment directed to MHC 1.

                • Reported results:

                                • No serious adverse events or decrements in neuropsychological tests.

                                • Average improvement of 13.2 points in total UPDRS (OFF) in ten patients at 6 months.

                                • Average improvement of 29 points (OFF) in 2 or two evaluable patients at 15 months.

                                • One patient had an improvement of >45 points in both ON and OFF scores.

 

 

 

(Hauser 1999, Obeso 1997)

Increase number of implanted cells ("dose" of tissue).

Increase number of surviving implanted cells.

Use trophic factors and trophic factor secreting cells, or sertoli cells, which may be genetically engineered to augment

    the viability of transplanted cells and to enhance neurotic extension.

Optimize the length and dosage of immunosuppressive drugs

Potentially stop prescription of Levodopa because it has shown toxicity to cultured DA neurons.

Attempt other sites of implantation.

Perform a controlled safety and efficacy trial in order to establish consistent and reproducible results.

Make progress toward quickening the beneficial responses of the transplantation.

Make progress toward eliminating all medications associated with the disease.

Attempt multiple needle passes through the brain.

Perform more immunological studies.

Make progress toward creating and obtaining the optimal donor cell:

                • Neural tissue will most likely be of animal origin so that there can be a imitless supply of readily available

                   cells and so that the majority of ethical issues are avoided.
 
                • Amitotic.

                • Homogenous and transmitter-specific.

                • Long-term anatomical and functional survival after transplantation.

                • Vascularized by a non-fenestrated capillary plexus.

                • Inexpensive.
 
 

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