Download lab meeting.ppt

Aim
• Examine the possibility of restoration of spiral
ganglion neurons by transplantation of fetal
neural stem cells (NSCs) into the modiolus of
cochlea.
Materials-donor
• Fetal mouse neural stem cells (NSCs) have the potential
for differentiation into neurons expressing green
fluorescence were used as donor cells.
• NSC spheres were obtained from the neuroepithelium of
the dorsal telencephalon of embryos at embryonic day
11.5 of C57BL/6JTg14 (Act-EGFP) Obs-Y01 transgenic mice
using the neurosphere culture medium .
• Secondary neurospheres, which exhibited expression of
nestin, but no expression of TuJ1 or GFAP, were collected,
dissociated and suspended for transplantation at a
density of 1×105 cells/ml in the neurosphere culture
medium.
Materials-recipient
• C57BL/6 mice at 6 weeks of age affected by
cisplatin, in which severe degeneration of
SGNs is induced.
• In this model, about 60% of SGNs disappear at
14 days after cisplatin treatment .
Surgery procedure
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•A cisplatin solution (2.5 mg/ml) was injected from the left
posterior semicircular canal.
The left cochlea of recipient animals was exposed 14 days after
deafening.
2 μl NSC was injected into the cochlea through the round
window toward the direction of the cochlear modiolus by
Hamilton syringe and infusion pump.
14 days after transplantation, the left cochlea was re-exposed
and 4% paraformaldehyde was gently perfused into the
perilymph from the round window.
The animals were then sacrificed. The temporal bones were
collected and immersed in the same fixative for 4 h at 4 ℃.
Cryostat sections 10 μm thick were made, and the midmodiolus sections were used for histological analysis.
Staining
• The cell fates of grafted NSCs were determined by
immunohistochemistry for Tuj 1 (marker for neurons) or GFAP
(marker of glial cells).
• Counterstaining with DAPI.
• The numbers of transplant-derived cells in the modiolus in one
section and the ratios of positivity for each marker in
transplant-derived cells were examined.
• Both EGFP- and DAPI-positive cells were defined as transplantderived cells. The average of two sections for each animal was
defined as the number for that animal. The ratio of positivity
for each marker in transplant-derived cells in the modiolus was
then calculated.
Result
• Robust survival of the injected
cells was found in cochleae.
• NSC-derived cells expressing
GFP located in the modiolus of
cochleae or scala tympani,
settled in the apical portion of
the modiolus .
• The mean and standard
deviation of numbers of NSC
derived cells in the modiolus
was 190.5 and 60.8.
• NSC-derived cells in the
modiolus exhibited
expression of TuJ1.
• TuJ1-positive grafted
cells were
predominantly located
in the osseous spiral
lamina or spiral ganglion
• Expression of TuJ1 was
observed in about 10%
of NSC-derived cells.
• NSC-derived cells
in the modiolus
exhibited
expression of
GFAP.
• GFAP-positive
grafted cells were
found in the
modiolus and
spiral ganglion.
TuJ1-positive grafted cells were positioned on the periphery
of grafted cells (Fig2A),
GFAP-positive grafted cells were mainly located in the center
of grafted cells (Fig. 2B).
Discussion
• NSCs injected in the basal portion of the
modiolus settled in the apical end of the
modiolus and osseous spiral lamina,
indicating the high potential of NSCs for
migration.
• NSC-derived cells differentiated into neurons
in the modiolus, indicating that NSC
transplantation into the modiolus may be
utilized for restoration of SGNs.
Discussion
• Promotion of the activity of neural
differentiation or increase of numbers of
surviving NSC-derived cells was crucial for
functional recovery of SGNs. -Neurotrophins
• The microenvironments where NSCs settled
influence the fate of grafted NSCs, Cytokines
associated with inflammation influence the
survival and differentiation of NSCs.
Conclusion
• Injection of NSCs into the modiolus of injured
cochleae results in robust survival of grafted
NSCs in the modiolus.
• Grafted NSCs differentiate into neurons in
the modiolus, although their number is
limited. NSC is a possible candidate of cell
therapy for restoration of SGNs.
My plan
•Examine the possibility of restoration of spiral
ganglion neurons and function reservation by
transplantation of human neuronal precursor
cells (NPCs) into the internal auditory canal
(IAC) thru cochleostomy in the Guinea Pig.
•Examine the morphology/function change of
the brain/contralateral cochlea after Ouabain
deafening. (The animals are hard to
anesthetize after Ouabain application. )
Materials
• Donor cells: Human NPCs
• Recipient: Hartley Guinea Pigs 2~3 weeks old
Procedure
1. 18 animals, 6 deafened with cell implantation,( 3 for 1w, 3 for
4w), 6 undeafened with cell implantation,(3 for 1w, 3 for 4w),
3 deafened without cell implantation for 4w, 3 undeafened
without cell implantation for 4w.
2. BL-clk ABR for both side of ear .
3. 10M Ouabain 5/10μl deafen left ear thru round window
membrane for 8 animals.
4. Post-deafening (2week) clk ABR for both ear.
5. 5 μl NPCs injection thru cochleostomy by Hamilton syringe
and pump.
6. Post-implant (1,2,4 week) clk ABR for both ear.
7. Sacrifice 1(4) week(s) and harvest both side of cochlea with
BS.
8. Staining.
Tips and Hurdles
•Accurate puncture into the IAC with little cochleostomy and
try to avoid the leakage of perilymph and CSF.
•Accurate volume for the NPCs is hard as the needle was
inside the cochlea and could not be show by microscope.
•Anesthetized by Xylazine & Ketamine is not strong enough to
do the the surgery as it would last for 2 hours or more, could
we use gas?
•How to care the immune action to the NPCs? Should
antibody and immune suppressor be applied?
•Is it available for click ABR to appraise the hearing fuction
after cell implantation?