U.S. patent application number 11/171876 was filed with the patent office on 2006-02-23 for retinal cell grafts and instrument for implanting.
Invention is credited to Stephen E. Hughes.
Application Number | 20060039993 11/171876 |
Document ID | / |
Family ID | 27014715 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060039993 |
Kind Code |
A1 |
Hughes; Stephen E. |
February 23, 2006 |
Retinal cell grafts and instrument for implanting
Abstract
Surgical instruments, surgical techniques, retinal cell grafts,
retinal cell and tissue isolation techniques, and a method for
transplanting retinal cells, including photoreceptors, and/or
retinal pigment epithelium, with the cells in the isolated tissue
having their normal cell to cell configuration are disclosed.
Inventors: |
Hughes; Stephen E.; (Delmar,
NY) |
Correspondence
Address: |
Daniel B. Schein, Ph.D.;Schein & Cai LLP
100 Century Center Ct.
San Jose
CA
95112
US
|
Family ID: |
27014715 |
Appl. No.: |
11/171876 |
Filed: |
June 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10323958 |
Dec 17, 2002 |
6955809 |
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11171876 |
Jun 29, 2005 |
|
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|
09411122 |
Oct 4, 1999 |
6579256 |
|
|
10323958 |
Dec 17, 2002 |
|
|
|
08322735 |
Oct 13, 1994 |
5962027 |
|
|
09411122 |
Oct 4, 1999 |
|
|
|
07566996 |
Aug 13, 1990 |
|
|
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08322735 |
Oct 13, 1994 |
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07394377 |
Aug 14, 1989 |
|
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|
07566996 |
Aug 13, 1990 |
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Current U.S.
Class: |
424/571 ;
604/500 |
Current CPC
Class: |
A61L 27/3891 20130101;
A61L 2430/16 20130101; A61K 35/44 20130101; A61B 90/00 20160201;
A61L 27/3641 20130101; A61L 27/3839 20130101; A61L 27/3683
20130101; A61L 27/3604 20130101; A61L 27/383 20130101; A61M
2025/0042 20130101; A61L 27/3813 20130101; A61F 9/007 20130101;
A61L 27/3804 20130101; A61F 9/0017 20130101; A61B 2017/00969
20130101; A61F 2/14 20130101 |
Class at
Publication: |
424/571 ;
604/500 |
International
Class: |
A61K 35/44 20060101
A61K035/44; A61M 31/00 20060101 A61M031/00 |
Claims
1. A retinal cell graft and instrument for implantation
combination, wherein said instrument can be used to implant said
retinal cell graft into a host eye, wherein, said graft comprises a
confluent population of cells from a donor eye, wherein said
population is isolated from the retina of the donor eye, said
population of cells comprising at least one of the group consisting
of photoreceptor cells, and retinal pigment epithelial cells, said
confluent population of cells having the same cell to cell
organization in the graft as in the donor eye.
2. The combination of claim 1, wherein said graft comprises
photoreceptor cells and retinal pigment epithelial cells.
3. The combination of claim 2, wherein said confluent population of
cells isolated from retina comprises a portion of the retina
lacking at least one layer of cells found in the intact retina
prior to isolation therefrom.
4. The combination of claim 1, wherein said cells are from an adult
donor.
5. The combination of claim 2, wherein said cells are from an adult
donor.
6. The combination of claim 1, wherein said instrument comprises:
an elongate tube having a distal end and a proximal end, said tube
having a lumen passing from said proximal end to said distal end,
said distal end having a distal tip, wherein said lumen forms an
opening in said tip, said instrument further comprising a plunger,
said plunger and tube being capable of relative sliding motion with
respect to each other, wherein the outer diameter of said distal
end of said elongate tube is sufficiently small to be inserted
through the pars plana area of an eye and the length of said distal
end of said elongate tube is sufficient to permit said opening in
said tip to be inserted beneath the retina through an opening
therein, wherein relative sliding motion of said plunger and said
tube can express solid or semisolid material within said lumen from
said lumen through said opening in said tip into the subretinal
space of an eye into which said tip is inserted.
7. The combination of claim 6, wherein said cells are from an adult
donor.
8. The combination of claim 6, wherein said tube may be retracted
with respect to said plunger to express said graft from said
tip.
9. An instrument for subretinal implantation of a solid or
semisolid therapeutic implant including a graft as recited in claim
1, wherein the therapeutic implant can be a retinal cell graft
comprising a confluent population of cells from a donor eye retina
and the population of cells comprises at least one of the group
consisting of photoreceptor cells, and retinal pigment epithelial
cells, and the confluent population of cells has the same cell to
cell organization in the graft as in the donor eye, said instrument
comprising: an elongate tube having a distal end and a proximal
end, said tube having a lumen passing from said proximal end to
said distal end, said distal end having a distal tip, wherein said
lumen forms an opening in said tip, said instrument further
comprising a plunger, said plunger and tube being capable of
relative sliding motion with respect to each other, wherein the
outer diameter of said distal end of said elongate tube is
sufficiently small to be inserted through the pars plana area of an
eye and the length of said distal end of said elongate tube is
sufficient to permit said opening in said tip to be inserted
beneath the retina through an opening therein, wherein relative
sliding motion of said plunger and said tube can express solid or
semisolid material within said lumen from said lumen through said
opening in said tip into the subretinal space of an eye into which
said tip is inserted.
10. The instrument of claim 9, further comprising a retinal cell
graft that can be implanted in the subretinal space of a host eye,
said retinal cell graft comprising a confluent population of cells
from a donor eye, wherein said population is isolated from a retina
and said population comprises at least one of the group consisting
of photoreceptor cells, and retinal pigment epithelial cells.
11. The instrument of claim 10, wherein said graft comprises a
layer of human photoreceptor cells and a layer of retinal pigment
epithelial cells.
12. The instrument of claim 10, wherein said confluent population
of cells comprises a portion of a human retina having at least one
layer of cells found in an intact retina missing therefrom.
13. The instrument of claim 10, wherein said cells are from an
adult donor.
14. The instrument of claim 11, wherein said cells are from an
adult donor.
15. The instrument of claim 9, wherein retraction of said tube with
respect to said plunger will express an implant within said tube
from said tip.
16. The instrument of claim 10, wherein retraction of said tube
with respect to said plunger will express an implant within said
tube from said tip.
17. The instrument of claim 11, wherein retraction of said tube
with respect to said plunger will express an implant within said
tube from said tip.
18. The instrument of claim 12, wherein retraction of said tube
with respect to said plunger will express an implant within said
tube from said tip.
19. The instrument of claim 13, wherein retraction of said tube
with respect to said plunger will express an implant within said
tube from said tip.
20. The instrument of claim 14, wherein retraction of said tube
with respect to said plunger will express an implant within said
tube from said tip.
Description
PRIORITY INFORMATION
[0001] This application is a divisional of Ser. No. 10/323,958,
filed Dec. 17, 2002, which is a divisional of Ser. No. 09/411,122
filed Oct. 4, 1999, now U.S. Pat. No. 6,579,256, which is a
divisional of Ser. No. 08/322,735, filed Oct. 13, 1994, now U.S.
Pat. No. 5,962,027, which is a continuation of Ser. No. 07/566,996
filed Aug. 13, 1990, abandoned, which is a continuation in part of
Ser. No. 07/394,377, filed Aug. 14, 1989, abandoned, all of which
are incorporated by reference as if reproduced in full below.
BACKGROUND OF THE INVENTION
[0002] The present invention relates in general to surgical
instruments, surgical techniques, and cell and tissue isolation
techniques. More particularly, the present invention is directed to
a surgical tool for transplanting retinal cells, epithelium and
choroidea within their normal planar configuration, a graft for
transplantation to the subretinal region of the eye, a method for
preparing such grafts for transplantation, and a method for
reconstructing dystrophic retinas, retinal pigment epithelial
layers and choroids.
[0003] The retina is the sensory epithelial surface that lines the
posterior aspect of the eye, receives the image formed by the lens,
transduces this image into neural impulses and conveys this
information to the brain by the optic nerve. The retina comprises a
number of layers, namely, the ganglion cell layer, inner plexiform
layer, inner nuclear layer, outer plexiform layer, outer nuclear
layer, photoreceptor inner segments and outer segments. The outer
nuclear layer comprises the cell bodies of the photoreceptor cells
with the inner and outer segments being extensions of the cell
bodies.
[0004] The choroid is a vascular membrane containing large branched
pigment cells that lies between the retina and the sclerotic coat
of the vertebrate eye. Immediately between the choroid and the
retina is the retinal pigment epithelium which forms an intimate
structural and functional relationship with the photoreceptor
cells.
[0005] Several forms of blindness are primarily related to the loss
of photoreceptor cells caused by defects in the retina, retinal
pigment epithelium, choroid or possibly other factors (e.g. intense
light, retinal detachment, intraocular bleeding). In several
retinal degenerative diseases select populations of cells are lost.
Specifically, in macular degeneration and retinitis pigmentosa
retinal photoreceptors degenerate while other cells in the retina
as well as the retina's central connections are maintained. In an
effort to recover what was previously thought to be an irreparably
injured retina, researchers have suggested various forms of grafts
and transplantation techniques, none of which constitute an
effective manner for reconstructing a dystrophic retina.
[0006] The transplantation of retinal cells to the eye can be
traced to a report by Royo et al., Growth 23: 313-336 (1959) in
which embryonic retina was transplanted to the anterior chamber of
the maternal eye. A variety of cells were reported to survive,
including photoreceptors. Subsequently del Cerro was able to repeat
and extend these experiments (del Cerro et al., Invest. Ophthalmol.
Vis. Sci. 26: 1182-1185, 1985). Soon afterward Turner, et al. Dev.
Brain Res. 26:91-104 (1986) showed that neonatal retinal tissue
could be transplanted into retinal wounds.
[0007] In related studies, Simmons et al., Soc. Neurosci. Abstr.
10: 668 (1984) demonstrated that embryonic retina could be
transplanted intracranially, survive, show considerable normal
development, be able to innervate central structures, and activate
these structures in a light-dependent fashion. Furthermore, these
intracranial transplants could elicit light-dependent behavioral
responses (pupillary reflex) that were mediated through the host's
nervous system. Klassen et al., Exp. Neurol. 102: 102-108 (1988)
and Klassen et al. Proc. Natl. Acad., Sci. USA 84:6958-6960
(1987).
[0008] Li and Turner, Exp. Eye Res. 47:911 (1988) have proposed the
transplantation of retinal pigment epithelium (RPE) into the
subretinal space as a therapeutic approach in the RCS dystrophic
rat to replace defective mutant RPE cells with their healthy
wild-type counterparts. According to their approach, RPE were
isolated from 6- to 8-day old black eyed rats and grafted into the
subretinal space by using a lesion paradigm which penetrates
through the sclera and choroid. A 1 .mu.l injection of RPE
(40,000-60,000 cells) was made at the incision site into the
subretinal space by means of a 10 .mu.l syringe to which was
attached a 30 gauge needle. However, this method destroys the
cellular polarity and native organization of the donor retinal
pigment epithelium which is desirable for transplants.
[0009] del Cerro, (del Cerro et al., Invest. Ophthalmol. Vis. Sci.
26: 1182-1185, 1985) reported a method for the transplantation of
tissue strips into the anterior chamber or into the host retina.
The strips were prepared by excising the neural retina from the
donor eye. The retina was then cut into suitable tissue strips
which were then injected into the appropriate location by means of
a 30 gauge needle or micropipette with the width of the strip
limited to the inner diameter of the needle (250 micrometers) and
the length of the strip being less than 1 millimeter. While del
Cerro reports that the intraocular transplantation of retinal
strips can survive, he notes that the procedure has some definite
limitations. For instance, his techniques do not allow for the
replacement of just the missing cells (e.g. photoreceptors) but
always include a mixture of retinal cells. Thus, with such a
transplant appropriate reconstruction of the dystrophic retina that
lacks a specific population of cells (e.g., photoreceptors) is not
possible.
[0010] del Cerro et al., Neurosci. Lett. 92: 21-26, 1988, also
reported a procedure for the transplantation of dissociated
neuroretinal cells. In this procedure, the donor retina is cut into
small pieces, incubated in trypsin for 15 minutes, and triturated
ii into a single cell suspension by aspirating it through a fine
pulled pipette. Comparable to the Li and Turner approach discussed
above, this procedure destroys the organized native structure of
the transplant, including the donor outer nuclear layer; the strict
organization of the photoreceptors with the outer segments directed
toward the pigment epithelium and the synaptic terminals facing the
outer plexiform layer are lost. Furthermore, no means of isolating
and purifying any given population of retinal cells (e.g.
photoreceptors) from other retinal cells was demonstrated.
[0011] It is believed by the present inventor that it is necessary
to maintain the photoreceptors in an organized outer nuclear layer
structure in order to restore a reasonable degree of vision. This
conclusion is based on the well known optical characteristics of
photoreceptors (outer segments act as light guides) and clinical
evidence showing that folds or similar, even minor disruptions in
the retinal geometry can severely degrade visual acuity.
SUMMARY OF THE INVENTION
[0012] Among the objects of the present invention, therefore, may
be noted the provision of a method for preparation of a graft for
use in the reconstruction of a dystrophic retina; the provision of
such a method which conserves relatively large expanses of the
tissue harvested from a donor eye; the provision of such a method
in which the polarity and organization of the cells at the time of
harvest are maintained in the graft; the provision of a graft for
use in the reconstruction of a dystrophic retina; the provision of
such a graft which facilitates regrowth of photoreceptor axons by
maintaining the polar organization of the photoreceptor and the
close proximity of their postsynaptic targets with the adjacent
outer plexiform layer upon transplantation; the provision of a
surgical tool for use in the transplantation method which allows
appropriate retinotopic positioning and which protects
photoreceptors or other grafted tissue from damage prior to and as
the surgical device is positioned in the eye; and the provision of
a method for transplantation of grafts to the subretinal area of an
eye.
[0013] Briefly, therefore, the present invention is directed to a
method for the preparation of a graft for transplantation to the
subretinal area of a host eye. The method comprises providing donor
tissue and harvesting from that tissue a population of cells
selected from retinal cells, epithelial tissue or choroidal tissue,
the population of cells having the same organization and cellular
polarity as is present in normal tissue of that type. The
population of cells is laminated to a non-toxic and flexible
composition which substantially dissolves at body temperature.
[0014] The present invention is further directed to a method for
the preparation of a graft comprising photoreceptor cell bodies for
transplantation to the subretinal area of a host eye. The method
comprises providing a donor retina containing a layer of
photoreceptor cell bodies. The layer of photoreceptor cell bodies
is isolated from at least one other layer of cells of the donor
retina in a manner that maintains the layer of photoreceptor cell
bodies in the same organization and cellular polarity as is present
in normal tissue of that type.
[0015] The present invention is further directed to a graft for
transplantation to the subretinal area of a host eye. The graft
comprises a laminate of a non-toxic and flexible composition which
substantially dissolves at body temperature and a population of
cells harvested from a donor eye, the population of cells being
selected from retinal cells, epithelial tissue and choroidal
tissue. The population of cells has the same organization and
cellular polarity as is present in normal tissue of that type.
[0016] The present invention is further directed to a graft for
transplantation to the subretinal area of a host eye. The graft
comprises a population of photoreceptor cell bodies harvested from
a donor retina, the population of photoreceptor cell bodies having
the same organization and cellular polarity as is present in normal
tissue of that type, the graft having an essential absence of at
least one layer of cells present in the donor retina.
[0017] The present invention is further directed to a method for
transplanting to the subretinal area of a host's eye a graft
comprising a population of cells. The method comprises providing a
graft comprising a population of cells selected from retinal cells
isolated from at least one other layer of cells within the retina,
epithelial tissue and choroidal tissue, the population of cells
being maintained in the same organization and t cellular polarity
as is present in normal tissue of that type. An incision is made
through the host's eye, the retina is at least partially detached
to permit access to the subretinal area and the graft is positioned
in the accessed subretinal area.
[0018] The present invention is further directed to an instrument
for the implantation of an intact planar cellular structure between
the retina and supporting tissues in an eye. The instrument
comprises an elongate supporting platform for holding the planar
cellular structure. The platform has a distal end for insertion
into an eye, and a proximal end. The distal edge of the platform is
convexly curved for facilitating the insertion of the platform into
the eye and the advancement of the platform between the retina and
the supporting tissues. The instrument has a side rail on each side
of the platform for retaining the planar cellular structure on the
platform, the distal ends of the side rails being rounded, and the
distal portions of the side rails tapering toward the distal end of
the platform to facilitate the insertion of the instrument between
the retina and the supporting tissue.
[0019] The present invention is further directed to an instrument
for the implantation of an intact planar cellular structure between
the retina and supporting tissues in an eye. The instrument
comprises an elongate tube, having a flat, wide cross-section, with
a top, a bottom for supporting the planar cellular structure, and
opposing sides. The tube has a beveled distal edge facilitating the
insertion of the tube into the eye and the advancement of the tube
between the retina and the supporting tissues. The instrument also
comprises plunger means for ejecting a planar cellular structure
from the distal end of the tube.
[0020] The present invention is further directed to a kit for
transplantation of a graft to the subretinal area of a host eye.
The kit contains a graft comprising a population of cells selected
from retinal cells, epithelial tissue and choroidal tissue, the
population of cells being maintained in the same organization and
cellular polarity as is present in normal tissue of that type. The
kit additionally contains a surgical instrument comprising an
elongate tube, having a flat, wide cross-section, with a top, a
bottom for supporting the planar cellular structure, and opposing
sides. The tube has a beveled distal edge facilitating the
insertion of the tube into the eye and the advancement of the tube
between the retina and the supporting tissues. The surgical
instrument also comprises plunger means for ejecting a planar
cellular structure from the distal end of the tube.
[0021] Other objects and features of the invention will be in part
apparent and in part pointed out hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a photograph of a cryostat section of normal rat
retina as set forth in Example 1;
[0023] FIG. 2 is a photograph of a blinded rat retina following
constant illumination as set forth in Example 1;
[0024] FIG. 3 is a schematic of a donor retina;
[0025] FIG. 4 is a schematic of a flattened retina;
[0026] FIG. 5 is a schematic of a flattened retina mounted to a
substrate;
[0027] FIG. 6 is a schematic of a sectioned retina mounted to a
substrate;
[0028] FIG. 7 is a schematic of a laminate comprising a retina
section on a supporting, stabilizing substrate;
[0029] FIG. 8 is a schematic top plan view of the laminate of FIG.
7, showing a graft (dashed lines) comprising a photoreceptor cell
layer and a supporting, stabilizing substrate;
[0030] FIG. 9 is a perspective view of a first embodiment of an
instrument adapted for implanting an intact planar cellular
structure between the retina and supporting tissues in an eye;
[0031] FIG. 10 is a side elevation view of a second embodiment of
an instrument adapted for implanting an intact planar cellular
structure between the retina and supporting tissues in an eye, with
the plunger in its retracted position, and with portions broken
away to show detail;
[0032] FIG. 11 is a top plan view of the instrument shown in FIG.
10;
[0033] FIG. 12 is a side elevation view of the instrument of the
second embodiment, with the plunger in its extended position, and
with portions broken away to show detail;
[0034] FIG. 13 is a top plan view of the instrument shown in FIG.
12;
[0035] FIG. 14 is a partial longitudinal cross-sectional view of
the instrument, showing part of a planar cellular structure loaded
therein;
[0036] FIG. 15 is a top plan view of a first alternative
construction of the second embodiment;
[0037] FIG. 16 is a top plan view of a second alternative
construction of the second embodiment;
[0038] FIG. 17 is a top plan view of a third alternative
construction of the second embodiment; and
[0039] FIG. 18 is a top plan view of the second embodiment, showing
an alternative plunger means.
[0040] FIG. 19 is a horizontal section through an eye illustrating
a trans-corneal surgical approach;
[0041] FIG. 20 is a horizontal section through an eye illustrating
a trans-choroidal and scleral surgical approach;
[0042] FIG. 21 is a photograph of transplanted photoreceptors as
set forth in Example 1;
[0043] FIG. 22 is a photograph of donor photoreceptors transplanted
at the posterior pole of the recipient eye as set forth in Example
1;
[0044] FIG. 23 shows the interface between the transplant and the
adjacent retina devoid of outer nuclear layer as set forth in
Example 1;
[0045] FIG. 24 is a photograph illustrating FITC fluorescent
micrograph of antibody Ret P-1 specific for opsin as set forth in
Example 1;
[0046] FIG. 25 is a series of photograph panels illustrating in A,
transplanted photoreceptors attached to recipient or host retina,
in B, fluorescent micrograph showing transplanted cells showing Dil
fluorescence, identifying them as transplanted tissue, and in C, a
micrograph illustrating FITC fluorescence of antibody specific for
opsin as set forth in Example 1;
[0047] FIG. 26 comprises two micrograph panels, in A, transplant of
nature rat photoreceptors to adult light damaged recipient or host,
and in B, transplant of human photoreceptor from adult donor to
adult light damaged rat host or recipient as set forth in Example
4;
[0048] FIGS. 27a, 27b, 27c, 27d are .sup.14C 2-deoxyglucose (2DG)
autoradiographs, DYST-dystrophic, TRANS-transplant as set forth in
Example 5; and
[0049] FIGS. 28a, 28b, 28c and 28d are a series of photographs
showing pupillary reflex to light as set forth in Example 9.
DETAILED DESCRIPTION
[0050] As used herein, the term "donor" shall mean the same or
different organism relative to the host and the term "donor tissue"
shall mean tissue harvested from the same or different organism
relative to the host.
[0051] Several forms of blindness such as retinitis pigmentosa,
retinal detachment, macular degeneration, and light
exposure-related blindness, are primarily related to the loss of
the photoreceptors in the eye. However, destruction of the
photoreceptors does not necessarily lead to the loss of the
remaining retina or axons that connect the retina to the brain.
Surprisingly, it has been discovered that some degree of vision can
be restored by replacing damaged photoreceptors with photoreceptors
harvested from a donor and which are maintained in their original
organization and cellular polarity.
[0052] FIG. 1 is a photograph of a cryostat section of normal rat
retina. FIG. 2 is a photograph of a cryostat section of a rat
retina following constant illumination which destroys the
photoreceptor (outer nuclear) layer while leaving other retinal
layers and cells largely intact. In these and subsequent figures,
the retina or layers thereof, e.g., the ganglion cell layer ("G"),
inner plexiform layer ("IPL"), inner nuclear layer ("INL"), outer
plexiform layer ("OPL"), outer nuclear layer ("ONL"), inner
segments ("IS"), outer segments ("OS"), and retinal pigment
epithelium ("RPE"), are shown, respectively, from top to
bottom.
[0053] Referring now to FIG. 3, a graft comprising photoreceptor
cells is prepared in accordance with a method of the present
invention by removing a donor retina 50 comprising inner retina
layers 52 and photoreceptor layer 54 from a donor eye. The donor
retina 50 is flattened (FIG. 4) by making a plurality of cuts
through the retina from locations near the center of the retina to
the outer edges thereof (see FIG. 8). Cuts can be made in other
directions if necessary.
[0054] As shown in FIG. 5, the flattened retina 56 is placed with
the photoreceptor side 54 down on a gelatin slab 58 which has been
surfaced so as to provide a flat surface 60 that is parallel to the
blade of a vibratome apparatus. The gelatin slab 58 is secured to a
conventional vibratome chuck of the vibratome apparatus. Molten
four to five per cent gelatin solution is deposited adjacent the
flattened retina/gelatin surface interface 61 and is drawn by
capillary action under the flattened retina causing the flattened
retina to float upon the gelatin slab 58. Excess molten gelatin is
promptly removed and the floating flattened retina is then cooled
to approximately 4.degree. C. with ice-cold Ringer's solution that
surrounds the gelatin block to cause the molten gelatin to gel and
hereby coat the bottom surface of the flattened retina and adhere
it to the gelatin block.
[0055] As shown in FIG. 6, the inner retina portion 52 is sectioned
from the top down at approximately 20 to 50 millimicrons until the
photoreceptor layer 54 is reached, thereby isolating the
photoreceptor layer from the inner layers of the retina, i.e., the
ganglion cell layer, inner plexiform layer, inner nuclear layer,
and outer plexiform layer. When the photoreceptor layer is reached,
the vibratome stage is advanced and a section from approximately
200 to 300 millimicrons thick obtained as shown in FIG. 7. The
thickness of this section is sufficient to undercut the
photoreceptor and form a laminate 62 consisting of a layer of
photoreceptor cells and the gelatin adhered thereto.
[0056] As shown in FIG. 8, the laminate 62 is cut vertically along
the dashed lines to create a graft 63 having a size appropriate for
transplantation. The surface of the graft should have a surface
area greater than about 1 square millimeter, preferably greater
than 2 square millimeters, and most preferably greater than 4
square millimeters or as large as may be practically handled. Thus
constructed, the graft may subtend a considerable extent of the
retinal surface.
[0057] The gelatin substrate adds mechanical strength and stability
to the easily damaged photoreceptor layer. As a result, the
flattened retinal tissue is less likely to be damaged and is more
easily manipulated during the transplantation procedure.
[0058] Gelatin is presently preferred as a substrate because of its
flexibility, apparent lack of toxicity to neural tissue and ability
to dissolve at body temperature.
[0059] However, other compositions such as ager or agarose which
also have the desirable characteristics of gelatin may be
substituted. Significantly, gelatin has not been found to interfere
with tissue growth or post-transplant interaction between the graft
and the underlying retinal pigment epithelium.
[0060] Gelatin is presently preferred as an adhesive to laminate
the retinal tissue to the substrate. However, other compositions,
including lectins such as concanavalin A, wheat germ agglutin, or
photoreactive reagents which gel or solidify upon exposure to light
and which also have the desirable characteristics of gelatin may be
substituted.
[0061] Advantageously, the gelatin or other substrate may
additionally serve as a carrier for any of a number of trophic
factors such as fibroblast growth factor, pharmacologic agents
including immunosuppressants such as cyclosporin A,
anti-inflamation agents such as dexamethasone, anti-angiogenic
factors, anti-glial agents, and anti-mitotic factors. Upon
dissolution of the substrate, the factor or agent becomes available
to impart the desired effect upon the surrounding tissue. The
dosage can be determined by established experimental techniques.
The substrate may contain biodegradable polymers to act as slow
release agents for pharmacologic substances that may be included in
the substrate.
[0062] The thickness of the graft comprising the sectioned
flattened retinal tissue and the substrate as discussed above is
only approximate and will vary as donor material varies. In
addition, sectioning may be facilitated and vibratome thickness
further calibrated from histological measurements of the thickness
of the retina, thereby providing further guides to sectioning
depth. Appropriate sectioning thicknesses or depth may be further
determined by microscopic examination and observation of the
sections.
[0063] As an alternative to mechanical, e.g., microtome,
sectioning, the donor retina may be chemically sectioned.
Specifically it is known that neurotoxic agents such as kainic acid
are toxic to cells in all retinal layers except the photoreceptor
layer (i.e., kainic acid does not damage photoreceptor cells).
Therefore, if the donor retina is treated with an appropriate
neurotoxic agent, the photoreceptor layer can be isolated. This
technique has the advantage of maintaining the retinal MUller cells
(which are not killed by kainic treatment) with the photoreceptor
cells. Since it is known that MUller cells help maintain
photoreceptor cells (both biochemically and structurally), the
isolation of MUller cells along with the photoreceptor cells could
be advantageous.
[0064] If desired, the graft may additionally contain the retinal
pigment epithelial cells. Because the RPE is tenuously adherent to
the retina, mechanical detachment of the retina from a donor eye
ordinarily will cause the RPE to separate from the retina and
remain attached to the choroid. However, through the use of
enzymatic techniques such as those described in Mayerson et al.,
Invest. Opthalmol. Vis, Sci. 25: 1599-1609, 1985, the retina can be
separated from the donor eye with the RPE attached. In accordance
with the present invention, grafts comprising the choroid may
additionally be prepared. To do so, the choroid is stripped off of
the scleral lining of the eye (with or without the RPE attached),
and flattened by making radial cuts. The donor choroid may then be
adhered to a substrate as previously described for the
photoreceptor cells and/or combined with a photoreceptor layer
which has been prepared as described above to form a laminate
comprising a photoreceptor layer adhered to a substrate, a RPE
layer and a choroidal layer.
[0065] Referring again to the Figures there is shown preferred
embodiments for the surgical instruments of this invention. The
surgical instruments are described in connection with a
photoreceptor isolation and transplantation method. The surgical
instruments and methods of this invention are particularly adapted
for isolation and transplantation of an intact sheet of cells from
a donor retina to a recipient retina and are characterized by the
maintenance of cell organization of the transplanted tissue
layer.
[0066] A first embodiment of an instrument for implanting an intact
planar cellular structure between the retina and supporting tissues
in an eye is indicated generally as 10 in FIG. 9. The instrument 10
may be made from acrylic, or some other suitable material that is
flexible and sterilizable. The instrument 10 comprises an elongate
platform 11 for holding the planar cellular structure. The platform
11 has a distal end 16 for insertion into the eye of the recipient,
and a proximal end 18. As shown and described herein the platform
11 is approximately 2 to 10 centimeters long, which is an
appropriate length for making implants in rodents and lower
primates. The platform 11 must be sufficiently long to extend into
the eye, between the retina and the supporting tissue, and thus
different platform lengths may be used, depending upon the
procedure being employed and upon the recipient. As shown and
described herein the platform is approximately 2.5 millimeters
wide, which is sufficiently wide for making implants in rodents and
lower primates. The platform 11 must be sufficiently wide to carry
and intact cellular structure for implanting, and thus different
platform widths may be used, depending upon the recipient.
[0067] As shown in FIG. 9, the edge 11a of the platform 11 at the
distal end 16 is preferably convexly curved to facilitate both the
insertion of the instrument 10 into the eye, and the advancement of
the instrument between the retina and the supporting tissue to
temporarily detach the retina, with a minimum of trauma. The
platform 11 is preferably concavely curved (with respect to the top
surface of the platform 11) along its longitudinal axis from the
distal end 16 to the proximal end 18. The curvature of the platform
11 facilitates the manipulation of the instrument 10 within the
eye, particularly the manipulation of the instrument between the
retina and the supporting tissue on the curved walls of the eye.
The radius of the curvature of the platform 11 will depend upon the
procedure and the recipient.
[0068] The platform 11 has side rails 12 and 14 on opposite sides
for retaining the planar cellular structure on the platform. As
shown in FIG. 9, the distal portions 12a and 14a of the side rails
taper from a point intermediate the distal and proximal ends of the
side rails toward their distal ends. The distal ends of the side
rails terminate in smoothly curved ends 12b and 14b, which are
proximal of the distal end 16 of the platform. The offset of the
distal ends of the rails, together with their rounded configuration
facilitates the insertion of the instrument into the eye and the
advancement of the instrument between the retina and the supporting
tissue. As shown and described herein, the proximal portions of the
side rails 12 and 14 are approximately 1 millimeter high, while the
distal portions 12a and 14a taper to about 0.5 millimeters. The
height of the side rails is made as small as possible, but they
must be slightly greater than the thickness of the planar cell
structure and the supporting substrate, and thus may vary depending
on the donor and the type of implantation being made (i.e., how
many cell layers are being implanted and thickness of the
substrate).
[0069] A second embodiment of an instrument for implanting an
intact planar cellular structure between the retina and supporting
tissues in an eye is indicated generally as 30 in FIGS. 10-14, and
18. The instrument 30 may be made from polyethylene, or some other
suitable material that is flexible and sterilizable. For example,
the instrument might be made of silicone rubber or silastic. The
instrument 30 comprises an elongate tube 32 having a flat, wide
cross-section, with a top 32a, a bottom 32b for supporting the
planar cellular structure, and opposing sides 32c and 32d. The tube
32 has a distal end 34 for insertion into the eye, and a proximal
end 36. The distal end 34 of the tube 32 is open for the discharge
of the planar cellular structure. The instrument 30 of the second
embodiment is preferable to the instrument 10 of the first
embodiment in at least one respect because the tube 32 has a top
32a which provides better protection for the planar cellular
structure to be implanted than the open platform 11.
[0070] As shown and described herein the tube 32 is approximately
3.5 centimeters long, which is an appropriate length for making
implants in rodents and lower primates. The tube 32 must be
sufficiently long to extend into the eye, between the retina and
the supporting tissue, and thus the different tube lengths may be
used, depending upon the procedure being employed and upon the
recipient. As shown and described herein the tube is approximately
2.5 centimeters wide, which is sufficiently wide for making
implants in rodents and lower primates. The tube must be
sufficiently wide to carry an intact cellular structure for
implanting, and thus different tube widths may be used, depending
upon the recipient. As shown and described herein, the sides 32c
and 32d are approximately 0.75 millimeters high. The height of the
sides is made as small as possible, but they must be slightly
greater than the thickness of the planar cell structure and
substrate, and thus may vary depending on the donor and the type of
implantation being made (i.e. how many cell layers are being
implanted and thickness of the substrate).
[0071] The distal end 34 of the tube can be beveled to facilitate
both the insertion of the tube into the eye, and the advancement of
the tube between the retina and the supporting tissues, with a
minimum of trauma. The end is preferably beveled at about
45.degree., from the top 32a to the bottom 32b. As shown in FIGS.
10 and 12, the distal end 34 of the tube 32 is also preferably
raked transversely across the tube (i.e. from side 32c to 32d)
toward the proximal end. The rake angle is preferably about
45.degree.. The raked distal end also facilitates the insertion of
the tube into the eye, and the advancement of the tube between the
retina and the supporting tissue. Moreover, raking the distal end
eliminates a sharp corner that could damage tissue.
[0072] The tube 32 is preferably concavely curved along its
longitudinal axis from the distal end 34 to the proximal end 36, so
that the top 32a is on the inside of the curve, and the bottom 32b
is on the outside of the curve. The curvature of the tube
facilitates the manipulation of the instrument 30 within the eye,
particularly the manipulation of the instrument between the retina
and the supporting tissue on the curved walls of the eye. The
radius of the curvature of the tube will depend on the procedure
and on the recipient.
[0073] The instrument 30 also comprises plunger means. As shown in
FIGS. 10-14, the plunger means is preferably a flat plunger 40
slidably received in the tube so that relative sliding motion
between the tube 32 and the plunger 40 urges a planar cellular
structure that is in the tube out the distal end of the tube. The
plunger 40 may be made of polymethylmethacrylate. The proximal end
of the plunger 40 projects a sufficient amount from the proximal
end of the tube 32 that the end of the plunger can be manipulated
even when the distal portion of the tube is in an eye. The
preferred method of operating the instrument 30 is that once the
distal end of the tube is properly located within the subretinal
area, the plunger 40 is held in place as the tube 32 is gradually
withdrawn to eject the cellular structure.
[0074] Alternatively, as shown in FIG. 18, the plunger means may
comprise means for applying hydraulic pressure on the contents of
the tube. In this case the proximal end 36 of the tube 32 is
connected to a line 41 connected to a source of fluid under
pressure. Fluid can be selectively supplied via the line 41 to the
proximal end of the tube, to displace the contents of the tube. The
fluid may be viscous, for example a 2% carboxymethylcellulose, or
non-viscous. Particularly in the later case, it may be desirable to
have a block 43 of gelatin or some other substance in the tube to
act as a mechanical plunger and to separate the fluid from the cell
structure being implanted. Gelatin is satisfactory because it is a
semi-solid, and because it will dissolve harmlessly if it is
ejected from the tube.
[0075] As shown in FIGS. 10-13, the instrument 30 preferably also
includes a lumen 42, extending generally parallel with the tube 32.
As used herein, lumen refers to any tube-like vessel, whether
separately provided or formed as a passageway in another structure.
The lumen 42 is attached to one of the sides of the tube 32, and
preferably side 32c so that the distal end of the tube rakes away
from the lumen. The lumen 42 has a distal end 44 generally adjacent
the distal end of the tube, and preferably slightly advanced
relative to the distal end of the tube. The proximal end 46 is
remote from the distal end, and may be provided with a connector 48
for connection with a source of fluid under pressure. Thus the
lumen 42 can eject a stream of fluid from its distal end 44 which
creates a fluid space ahead of the instrument, which helps separate
or detach the retina from the supporting tissue as the instrument
is advanced. The fluid may be a saline solution, or some other
fluid that will not harm the delicate eye tissues. Various
substances, such as anti-oxidants, anti-inflammatories,
anti-mitotic agents and local anesthetics can be provided in the
fluid for treatment of the eye or implanted tissue.
[0076] The raked distal end of the tube 32 follows generally in the
path opened by the fluid, thus minimizing direct contact of the
instrument and the eye tissue. The distal end of the lumen may be
beveled to facilitate the advancement of the instrument,
particularly at times when fluid is not being ejected from the
lumen. The end is preferably beveled at about 45.degree.. Of
course, rather than provide a separate lumen 42, the lumen could be
formed integrally in the walls of the tube 32.
[0077] A first alternate construction of instrument 30 is indicated
as 30A in FIG. 15. The instrument 30A is very similar in
construction to instrument 30, and corresponding parts are
identified with corresponding reference numerals. However, unlike
instrument 30, the instrument 30A includes a fiber optic filament
64 extending generally parallel with lumen 42, and positioned
between the lumen 42 and the tube 32. The fiber optic filament 64
facilitates the manipulation of the instrument and the proper
placement of the implant in two ways: a light source can be
provided at the proximal end of the fiber optic filament so that
the filament provides light at the distal end of the instrument, to
facilitate the visual observation procedure through the pupil.
Alternatively, a lens could be provided at the proximal end of the
fiber optic filament so that the filament can also be used for
direct observation at the distal end of the instrument.
[0078] Additionally, the fiber optic filament could allow for
laser-light cautery to control subretinal bleeding. Of course,
rather than provide a separate fiber optic filament 64, fiber optic
filaments could be incorporated into the walls of the tube 32 or
the lumen 42.
[0079] A second alternative construction of instrument 30 is
indicated as 30B in FIG. 16. The instrument 30B is very similar in
construction to instrument 30, and corresponding parts are
identified with corresponding reference numerals. However, unlike
instrument 30, the instrument 30B includes a lumen 66 extending
generally parallel with lumen 42, and positioned between the lumen
42 and the tube 32. The lumen 66 allows for the aspiration of
material from the distal end of the instrument. The proximal end of
the lumen 66 can be connected to a source of suction, to remove
excess fluid and debris. It is possible to incorporate the lumen 66
into the wall of the tube 32.
[0080] A third alternative construction of the instrument 30 is
indicated as 30C in FIG. 17. The instrument 30C is very similar in
construction to instrument 30, and corresponding parts are
identified with corresponding reference numerals. However, unlike
instrument 30, the instrument 30C includes a pair of lead wires 65,
terminating in an electrode 67 at their distal ends. The electrode
67 allows for cauterization of blood vessels. The proximal ends of
the leads 65 can be connected to a source of electrical power to
seal broken blood vessels. It is possible to incorporate the leads
65 into the wall of the tube 32.
[0081] Of course, two or more of the features described with
respect to the alternate embodiments 30A, 30B, and 30C could be
combined, if desired.
[0082] To transplant the retinal cells, including photoreceptors,
the host eye is prepared so as to reduce bleeding and surgical
trauma. A transcorneal surgical approach to the subretinal space is
one such approach and it will be understood that other surgical
approaches, such as transscleral and choroidal may also be used.
The preferred surgical approach in the rodent, FIG. 19, includes
making a transverse incision 70 in a cornea 72 of sufficient size
so as to allow insertion of a surgical instrument illustrated
schematically by reference characters 10 or 30. The instrument 10
is advanced under the iris, through the cornea 72 and to the ora
serrata 74 as illustrated in FIG. 19. The iris should be dilated
for example, with topical atropine. When the instrument 10 is used,
it detaches the retina as it is advanced under the retina and into
the sub-retinal space to the posterior pole 76 of the eye.
[0083] The channel defined by the side rails 12, 14 and the
intermediate cell supporting platform provides for the graft
comprising a photoreceptor layer 54 attached to the gelatin
substrate to be placed on the instrument 10 and guided into the
sub-retinal space, preferably with forceps or other suitable
instruments. After positioning the photoreceptor layer at the
desired transplant site, the gelatin is held in position with the
forceps while the carrier is removed. The edges of the corneal
incision are abutted after removal of the forceps to allow rapid,
sutureless healing. The eye should be patched during recovery.
[0084] If the surgical instrument 30 (FIGS. 10-14, and 18) is used
instead of the instrument 10, the graft comprising intact generally
planar sheet 54 of donor photoreceptors attached to the gelatin
substrate 62 is drawn into the elongate tube 32. The instrument 30
is then inserted through an appropriate sized incision in the
cornea and advanced under the iris. The iris will have been
dilated, for example, with topical atropine. The instrument 30 is
advanced to the ora serrata 74 of the host eye. If the instrument
30 includes a lumen 42, the retina is detached by the gentle force
of a perfusate such as a saline-like fluid, carboxymethylcellulose,
or 1-2% hyluronic acid ejected from the lumen 42. Advantageously,
the fluid may additionally contain anti-oxidants, anti-inflammation
agents, anesthetics or agents that slow the metabolic demand of the
host retina.
[0085] If the instrument 30 does not include a lumen 42, the retina
is detached by the walls of the surgical instrument as it is
advanced under the retina and into the subretinal space to the
posterior pole 76 of the eye. The graft comprising a photoreceptor
layer attached to the gelatin substrate is then transplanted by
moving the tube 32 in a direction away from the eye while keeping
the plunger 40 stationary. The plunger 40 is carefully withdrawn
out of the eye and the edges of the corneal incision are abutted
after removal to allow rapid, sutureless healing. Retinal
reattachment occurs rapidly and the photoreceptor sheet is held in
place in a sandwich-like arrangement between the retina and the
underlying eye tissues. The incision may require suturing.
[0086] FIG. 20 depicts a trans-choroidal and scleral surgical
approach as an alternative to the transcorneal approached described
above. Except for the point of entry, the surgical technique is
essentially the same as outlined above. Nevertheless, the
transcorneal approach is preferred because it has been found to
reduce bleeding and surgical trauma.
[0087] A further surgical approach is to diathermize in the pars
plana region to eliminate bleeding. The sclera is then incised and
the choroidal and epithelial tissue is diathermized. The surgical
tool is then inserted through the incision, the retina is
intercepted at the ora serrata and the graft is deposited in the
subretinal area otherwise as outlined elsewhere herein.
[0088] In yet a further surgical approach, entry is gained through
the pars plana area as outlined above and an incision is made in
the retina adjacent to the retinal macula. The surgical tool is
then inserted through the retinotomy and into the macular area.
[0089] It is known that the retina does not necessarily undergo
glial scar formation when it is damaged, unlike the adult central
nervous system as disclosed by Bigami et al., Exp. Eye Res.
28:63-69, (1979), and McConnel et al., Brain Res. 241:362-365
(1982). McConnel et al. suggest that this characteristic lack of
scar tissue contribute to a potential of retinal cells to regrow
severed axons within the eye.
[0090] In accordance with the present invention, it has recognized
that regrowth of photoreceptor axons may be facilitated by the
proximity of the post-synaptic targets of the photoreceptor within
the adjacent outer plexiform layer. In addition, growth across
substantial intervening neural or glial scar tissue is not
necessary in order for transplanted photoreceptors to make
appropriate connections with the recipient retina including neural
connections.
[0091] The following examples illustrates the invention.
EXAMPLE 1
Experimental Animals
[0092] Adult albino rats (Sprague-Dawley) were exposed to constant
illumination averaging 1900 lux for 2 to 4 weeks as described in
O'Steen, Exp. Neurol. 27:194 (1970).
[0093] As shown in FIG. 1, this exposure destroys most
photoreceptors, eliminating cells of the outer nuclear layer but
leaving the remaining neural retina intact. Photoreceptors for
transplantation were taken from 8-day-old normal rats of the same
strain that had been maintained under colony room illumination
(10-20 lux) on a 12 hr/12 hr light/dark cycle. Experimental animals
were anesthesized with ketamine and sodium pentobarbital. A
preoperative dose of dexamethasone (10 mg/kg IP) was also
administered.
Photoreceptor Preparation
[0094] The retina from the anesthetized 8-day-old rat was removed,
flattened with radial cuts and placed with the receptor side down
on a gelatin slab secured to the vibratome chuck. Molten gelatin
(4-5% solution) was deposited adjacent the retina at the
retina/gelatin interface and then cooled to 4.degree. C. with
ice-cold Ringer's solution. The retina was sectioned at 20 to 50
.mu.m until the photoreceptor layer was reached. When the
photoreceptor layer was reached, the stage was advanced and a thick
(200 to 300 .mu.m) section was taken, undercutting the
photoreceptor layer secured to the gelatin base.
Dil Labeling
[0095] The isolated outer nuclear layer was cultured overnight with
40 .mu.g/ml of dil
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
in Earle's MEM containing 10% fetal calf serum, incubated under
95%/5% oxygen/carbon dioxide mixture at room temperature. Labeling
techniques and fluorescent microscopy were otherwise as outlined by
Honig et al., J. Cell. Biol. 103:17, 1986. Dil was removed from
sections that were to be counterstained with FITC-labeled RET-P1
opsin antibody by prior washing in acetone.
Surgical Procedure
[0096] A transverse incision was made in the cornea sufficient to
allow insertion of a surgical instrument 10 that is 2.5 mm wide
with side rails 0.5 mm high or surgical instrument 30. The
instrument was advanced under the iris (dilated with topical
atropine) to the ora serrata, detaching the retina. The carrier was
then advanced under the retina into the subretinal space to the
posterior pole of the eye. The instrument allowed a graft
comprising a piece of the photoreceptor layer attached to the
gelatin substrate (up to 2.5.times.4 mm) to be guided into the
retinal space by fine forceps. The instrument was then removed
while the gelatin was held in position by the forceps. Following
removal of the forceps, the edges of the corneal incision were
abutted to allow rapid, sutureless healing. The eye was patched
during recovery and a prophylactic dose of penicillin was
administered. Upon removal of the patch, a veterinary ophthalmic
antibiotic ointment was applied.
[0097] Transplant recipients were maintained on a 12 hr/12 hr
light/dark cycle with an average light intensity of 50 lux.
Following appropriate survival times, the animal was overdosed with
pentobarbital and perfused transcardially with phosphate buffered
3% paraformaldehyde-2% gluteraldehyde solution. Cryostat sections
of both the light-blinded eye (control) and the eye receiving the
photoreceptor transplant were then cut (20 .mu.m).
Immunohistochemistry
[0098] Antibody labeling for opsin was performed on retinas fixed
with 3% paraformaldehyde and cryosectioned at 20 .mu.m.
Immunohistocheinical methods were otherwise as described in Hicks,
et al., J. Histochem Cytochem 35:1317 (1987). Elimination of the
primary antibody eliminated specific labeling for opsin.
Cryostat Sections
[0099] Cryostat sections made at 4 weeks post-transplantation are
shown in FIGS. 22-24. FIG. 22 is a low-power photomicrograph
showing the location of the photoreceptor transplant (between
arrowheads) at the posterior pole of the host eye Bar=0.5 mm. FIG.
23 is a higher-power photomicrograph showing the interface between
the transplant and the adjacent retina devoid of outer nuclear
layer. Arrows indicate the extent of the transplant (T). Arrowheads
indicate three possible residual photoreceptors that survived
constant illumination. Note their fusiform shape contrasts with the
rounder, more normal shape of the transplanted photoreceptors. H
& E stain. Bar=100 .mu.m. FIG. 24 is a FITC fluorescent
micrograph of antibody Ret P-1 specific for opsin in a section
adjacent to that shown in FIG. 23. Arrows indicate the extent of
the transplant. Transplanted cells are labeled for opsin indicating
they are photoreceptors. Some nonspecific fluorescence is evident
adjacent to the transplant. Bar=100 .mu.m.
[0100] Cryostat sections made at 3 weeks post-transplantation are
shown in FIGS. 25A-C. FIG. 25A is a H & E-stained
photomicrograph of a transplant and host retina. Note the
cell-sparse layer that resembles the outer plexiform layer
interposed between the host retina and the transplant. FIG. 25B is
a dil fluorescent photomicrograph of adjacent section. Transplanted
photoreceptors show dil fluorescence, identifying them as donor
tissue. FIG. 25C is a FITC fluorescent micrograph of antibody
RET-P1 in a section adjacent to that shown in FIG. 25A.
Transplanted cells are labeled for opsin, indicating that they are
photoreceptors. Bar=50 .mu.m.
Results
[0101] By using the transcorneal approach, it was found that the
positioning of the photoreceptor layer between the host's retina
and the adjacent epithelial and choroidal tissue layers of the eye
could be accomplished while minimizing the vascular damage and
subsequent bleeding into the eye. In addition, it was found that
this approach does not appear to disrupt the integrity of the
retina, which reattaches to the back of the eye with the
transplanted photoreceptors interposed between the retina and the
RPE. As shown in FIGS. 21-24, retinal reattachment appears to be
facilitated in the immediate area of the transplant. (The "T"
indicates transplanted sheets of photoreceptor cells). Using this
insertion method, it was possible to position the photoreceptors at
the posterior pole of the retina (FIG. 22).
[0102] To determine the viability of the transplanted
photoreceptors, cryostat sections (20 .mu.m) were made from both
the blinded eye (control) and the eye receiving the photoreceptor
transplant at 2, 4, or 6 weeks after transplantation. It was found
that the photoreceptors survived transplantation at all times
tested (36 out of 54 transplants). In most instances, the surviving
transplant approximated its size at the time of transplantation.
More importantly, there was no apparent reduction in transplant
size with longer survival times, suggesting that the transplants
were stable.
[0103] As a control, the contralateral eyes that did not receive a
photoreceptor transplant were examined. In these eyes, the retinas
possessed very few residual photoreceptors located adjacent to the
outer plexiform layer and the RPE. However, these residual cells
were abnormal in their appearance, having flattened, pyknotic cell
bodies instead of the rounded cell bodies of normal photoreceptors.
Furthermore, the residual photoreceptors did not form an outer
nuclear layer composed of columnarly stacked cell bodies, but
instead were found in isolation, or at most appear as a single or
double layer of cells (see FIG. 23) located mainly in the
peripheral retina.
[0104] The transplanted cells were easily distinguished from the
residual photoreceptors by a number of parameters. First, they were
found in discrete patches and have the characteristic columnar
stacking arrangement of up to about 12 cell bodies that is
characteristic of photoreceptor cells in the outer nuclear layer of
the normal retina. They did not have the flattened appearance of
the residual native photoreceptors, but instead have the round,
nonpyknotic cell body typical of normal transplanted cells.
Furthermore, the transplanted photoreceptors can form rosette
configurations, a characteristic of transplanted and cultured
retina while residual photoreceptors were not found in these
configurations.
[0105] To eliminate the possibility that the surgical procedure in
some manner induced the regeneration of native photoreceptors, sham
operations were performed. All procedures were performed as with
the photoreceptor transplants except that no photoreceptors were
attached to the inserted gelatin slab. While the retina reattached
to the back of the eye, in no instance were patches of
photoreceptors found.
[0106] To positively identify the photoreceptor patches in
experimental animals as transplanted tissue, the donor outer
nuclear layer was labeled with the fluorescent marker dil prior to
the transplantation. As shown in FIG. 25B, the photoreceptor
patches were fluorescently labeled while the host retina did not
show dil fluorescence.
[0107] To confirm that the transplants consisted of photoreceptors,
a monoclonal antibody specific for opsin, RET-P1 was used. As opsin
is found only in photoreceptors, any cell showing labeling for
opsin was, therefore, identified as a photoreceptor. As can be seen
in FIGS. 24 and 25C, the transplanted cells stain intensely for
opsin whereas other retinal cells are unstained. Positive staining
for opsin not only identifies these cells as photoreceptors but
indicates that these cells are still capable of producing the
protein moiety of visual pigment. Retina adjacent to the region of
the transplant shows only a few isolated photoreceptor cell bodies
(FIG. 23) that do not stain for opsin (FIG. 24) suggesting that
they are cones. Their lack of opsin staining, as well as their
location and appearance in H & E-stained material, confirms
that these cells are the host's residual photoreceptors (FIG.
23).
[0108] Harvesting the photoreceptor layer from the neonatal retina
does not appear to disrupt tissue organization. Once transplanted,
the photoreceptor layer maintained its characteristic columnar
arrangement of cell bodies for all survival times examined, thus
forming a new outer nuclear layer within the host's retina. In some
cases, strict polarity was lost and the rosettes were formed. By
light microscopy, the new layer appeared to be attached to the
host's outerplexiform layer (FIGS. 22 and 25A). This layer normally
is the site of synaptic contact between the photoreceptors and the
retina.
EXAMPLE 2
[0109] The procedures of Example 1 were repeated except as noted.
Substituted for the Sprague-Dawley rats were the rd mouse and the
RCS rat which are afflicted with inherited retinal degeneration. In
the rd mouse it is thought that the deficit resides in the
photoreceptor whereas in the RCS it is thought that the deficit
resides in the pigment epithelium. In these animals, almost all
photoreceptors are eliminated while the remaining retina is
preserved; either the rd mouse or the RCS rat were blinded by
constant illumination as set forth in Example 1. The rd mouse and
the RCS respectively received transplants of immature (7-8 day old
mouse or rat) and mature rat photoreceptors.
rd Mouse
[0110] The transplantation technique was adapted to the smaller
size of the mouse eye. This modification allowed sheets of intact
outernuclear layer to be transplanted to the subretinal space of
the mouse eye. Neonatal (8 days old) photoreceptors were
transplanted from rd control mice to the subretinal space of adult
rd mice. Survival times were for 2 weeks to 3 months. At all
survival times, it was found that the transplanted photoreceptors
survived, becoming physically attached to the outer portion of the
host retina and stained positive for opsin. In addition, the host
retina became reattached to the pigment epithelium.
[0111] In the rd mouse almost all photoreceptors are eliminated by
day 21. It was found that photoreceptors from a non-dystrophic
congenic control mouse can be transplanted to their appropriate
site within the adult rd mouse eye that lacks photoreceptors. These
transplanted photoreceptors were found to survive for as long as
tested (3 months). This length of time is significant since
photoreceptors of the rd mouse show signs of degeneration after
about 2 weeks and are almost completely eliminated after 3 weeks.
The survival of transplanted photoreceptors from congenic normal
donors to the adult rd mouse within the rd mouse supports findings
that indicate that the deficit within the rd mouse which causes the
degeneration of photoreceptors is endogenous to the rd
photoreceptors themselves.
RCS Rat
[0112] Photoreceptors from 7 to 8 day old RCS controls (normal)
were transplanted to the subretinal space in the eye of adult (3
month old) RCS rats. A two month survival period was allowed
because in this time period almost all host photoreceptors
degenerate in the RCS rat. It was found that the grafted.
photoreceptors survive transplantation to the subretinal space of
the RCS rat and show histotypic as well as immunological
characteristics of normal photoreceptors. In addition, it was found
that transplanted photoreceptors survive within their homotopic
location in the RCS rat whereas the RCS's own photoreceptors do
not.
Results
[0113] While it has been found that the transplanted photoreceptors
survive, produce opsin, and apparently integrate with the recipient
retina, they do not appear completely normal in that the number of
outer segments is reduced. However, photoreceptors lacking outer
segments are still capable of phototransduction as indicated in Pu
et al., J. Neorosci., 4:1559-1576, 1984. The relative scarcity of
outer segments has also been noted in retina transplanted to the
tecum. These retina have been shown to be functional as indicated
in Simon et al., Soc. Neorosci. Abstr., 10:668, (1984).
[0114] Conventional reasoning attributes the observed deficiency in
outer segments to be the possible consequence of the lack of
appropriate apposition of the RPE to the photoreceptors as
indicated in LaVail et al., (1971), noted above. However, it has
been found that RPE is present and in apparently normal apposition
to the photoreceptors, thus, the scarcity of outer segments here
would not appear to be related to inadequate contact between
photoreceptor and RPE. The failure of outer segment growth in the
presence of photoreceptor apposition to the RPE has also been seen
following retinal reattachment as reported by Anderson et al.,
Invest Opthalmol. Vis, Sci. 24:906-926, 1983.
EXAMPLE 3
[0115] The procedures of Example 1 were repeated except as
noted.
[0116] Donor photoreceptors were originally harvested at the
earliest ontogenetic time in which the photoreceptors could be
isolated from other portions of the retina (7-8 days old) since it
is generally believed that more embryonic and undifferentiated
neural tissue survives transplantation far better than more mature
and differentiated tissue. To determine the effect of developmental
age on photoreceptor survival and ability to integrate with the
host retina, photoreceptors were subsequently transplanted from 8,
9, 12, 15 and 30 day old rats into light damaged adults. These show
progressive development and maturation of the photoreceptors
including mature outer segments (at 15 and 30 days).
[0117] Using the same criteria as in Example 1, it was found that
for all ages tested the transplants survived for as long as
examined (2 months) and integrated with the host retina. FIG. 26,
panel A, is a photograph of a transplant of mature photoreceptors
(30 day old donor) to adult light damaged host. (T, Transplant).
120X. These observations suggest that photoreceptors have
characteristics that differ from other neural tissue that permits
them to be transplanted when they are essentially mature while
other neural tissue must be at a very immature stage for successful
transplantation to occur.
EXAMPLE 4
[0118] The procedures of Example 1 were repeated except as noted.
Photoreceptors were taken from the retina of donated human eyes
(obtained from the Missouri Lions and St. Louis Eye Banks)
following corneal removal. A portion of the retinas were tested for
viability by dye exclusion with trypan blue and didansyl cystine
staining. The photoreceptors excluded dye and appeared to be in
good condition. Hosts were adult albino rats (immune-suppressed
with cyclosporin A or immune-competent) exposed to constant
illumination.
[0119] With immune-suppression successful transplants were seen at
all survival times so far examined (one and two weeks; five of nine
cases), showing apparent physical integration with the host retina
and maintaining morphological features of the outer nuclear layer
as illustrated in FIG. 26B which shows a transplant of human
photoreceptors from adult donor to adult light damaged rat host.
(T, transplant). 120X.
[0120] The transplants stained positive for antiopsin antibody
RET-P1, identifying the transplanted cells as photoreceptors and
further indicating that they are still capable of producing visual
pigment. In contrast, transplants to immune-competent hosts showed
signs of rejection within one week of transplantation. Sham
operated animals showed no repopulation of the host retina with
photoreceptors.
[0121] The procedures of Example 1 were repeated except as
noted.
[0122] The 2DG functional mapping technique developed by Sokoloff
et al., J. Neurochem. 28:897-916 (1977) allows the measurement of
the relative levels of neural activity for a given stimulus
condition. For this reason, the 2DG technique appeared to be an
appropriate method of assessing the functional characteristics of
the transplant and its ability to activate the light damaged
retina.
[0123] Accordingly, patterns of 2DG uptake in the normal retina
were compared to that seen in the light-damaged retina, with and
without a photoreceptor transplant. These comparisons were made
under two different visual stimulus conditions: 1) darkness and 2)
strobe flicker at 10z. FIG. 27 illustrates the results of these
comparisons. H&E stained retina with corresponding
2-deoxyglucose autoradiographs. A and B normal retina. Sections cut
slightly tangentially to expand retinal layers. C. Dystrophic
(light-damaged) retina plus photoreceptor transplant (T) left of
arrow. Black and white lines at left on 2DG autoradiograph bracket
lower 2DG uptake in inner plexiform and ganglion cell layers. D.
Dystrophic retina plus photoreceptor transplant left of arrow. ONL;
outer nuclear layer, T; transplant, DYST; dystrophic, Bar=0.5
mm.
[0124] As shown in panel 27A, in darkness 2DG was preferentially
taken up in the outer portion of normal retina (photoreceptors and
possibly the inner nuclear layer). As shown in panel 27B, with
strobe flicker stimulation 2DG uptake extends through the thickness
of the normal retina. These patterns of 2DG uptake are in good
agreement with the known physiological characteristics of the
retina.
[0125] The outer retina might be expected to show high 2DG uptake
in the dark since photoreceptors, horizontal and some bipolar cells
are maximally depolarized in this situation. As strobe flicker is a
strong stimulus for the retina including the amacrine and retinal
ganglion cells, 2DG uptake across the entire retina is also to be
expected. It therefore appears that the 2DG uptake pattern in
normal retina reflects relative degrees of neural activity or
neural depolarization, and therefore is a useful indicator of
neural activity in the retina as it is in other areas of the
nervous system.
[0126] In the light-damaged retina which received a photoreceptor
transplant, the pattern of 2DG uptake was also dependent on the
stimulus conditions. In the dark, preferential uptake of 2DG was
limited to the photoreceptor transplant and the adjacent host inner
nuclear layer while relatively lower uptake was present in the
host's inner plexiform and ganglion cell layers. However, in the
strobe flicker condition, high 2DG uptake is present in the
transplant and, in addition, extended through the thickness of the
host's retina--but only in the area of the photoreceptor graft
(FIG. 27D). Adjacent host retina which did not receive the
photoreceptor transplant shows relatively low 2DG uptake.
[0127] In darkness, both the normal retina and the light-damaged
retina receiving the photoreceptor transplant show relatively high
uptake of 2DG in the photoreceptor and inner nuclear layers. The
similarity in the relative uptake patterns between these cases
suggests that the transplanted photoreceptors may have similar
functional characteristics as normal photoreceptors (i.e., they
depolarize in the dark and are capable of inducing a sustained
depolarization of some cells in the host's inner nuclear
layer).
[0128] In strobe flicker, the light-damaged retina receiving the
photoreceptor transplant showed high 2DG uptake through the entire
thickness of the retina much like that seen in the normal retina
under the same stimulus condition. Adjacent light-damaged retina
that did not receive a photoreceptor transplant showed relatively
low 2DG uptake. These comparisons show that the pattern of 2DG
uptake in the light-damaged retina approximates that seen in the
normal retina only in areas of the host retina that received
photoreceptor grafts. Adjacent areas of the host retina show
relatively low levels of 2DG uptake in both stimulus conditions.
The similarity in the 2DG uptake patterns between the light-damaged
retina following photoreceptor grafting and the normal retina in
both stimulus conditions suggests that the photoreceptor transplant
is capable of light-dependent activation of the light-damaged
retina.
EXAMPLE 6
[0129] The procedures of Example 1 were repeated except as
noted.
[0130] While activation of the host retina by the transplanted
photoreceptors was seen with deoxyglucose mapping the nature of
this activation was unclear. Specifically, does such activation
represent a nonsynaptic modulation of neurotransmitter release by
the transplanted photoreceptors or are the transplanted cells
forming synapses with elements of the host retina?
[0131] To address this issue, the ultrastructure of the
reconstructed retina was investigated. Following appropriate
survival times, animals were euthanized by overdose and immediately
enucleated. Control and experimental eyes for light microscopy were
fixed overnight in Bouin's solution. Following dehydration and
clearing, the tissue was embedded in paraffin. Sections were cut on
a rotary microtome. Eyes destined for plastic embedment were fixed
for 2 hours in buffered 2.5% glutaraldehyde. After 1/2 hour of
aldehyde fixation, the anterior segment and lens were removed to
facilitate penetration of the fixative. Following primary fixation,
eyes for light microscopy were processed further for methacrylate
embedding. Two to 5 .mu.m sections were cut on a rotary microtome
using glass "Ralph" knives. In eyes for ultrastructural analysis,
the area receiving the transplant was localized using the Dil label
and excess tissue was trimmed before osmium posifixation. Following
dehydration and clearing, the tissue was embedded in Epon/Araldite
(Mollenhauer, 1964). Blocks were surveyed by staining semithin
sections with toluidine blue. When the transplant was located, thin
sections were cut and stained with uranyl acetate and lead citrate
for examination on the EM.
[0132] A new outer plexiform-like layer was visible at the
interface of the transplanted ONL and the host inner nuclear layer.
Ribbon synapses were evident within this OPL. These synapses are
characteristic of those formed by rod photoreceptors, with an
electron dense ribbon surrounded by a cluster of vesicles. Ribbon
synapses are found only rarely in control light-damaged retina. In
addition to ribbon synapses, the transplanted photoreceptors also
display inner segments, connecting cilia, and outer segment
membranes. These results suggest that synaptic connections between
transplanted photoreceptors and host cells were made indicating
that the light-dependent activation may, at least in part, be
synaptically mediated.
EXAMPLE 7
[0133] The procedures of Example 1 were repeated except as
noted.
[0134] The functional capabilities of the transplanted
photoreceptors and reconstructed retina was ascertained by
recording visually evoked cortical potentials ("VEP"). To record
the VEP, animals were implanted with stainless-steel screw
electrodes embedded in the skull. The active electrodes were placed
2 mm anterior to lambda (bilaterally), and referred to a second
electrode placed anterior to bregma. Both electrodes were placed 2
mm lateral to the midline and positioned on the dura. A third screw
was placed above the nasal cavity to serve as a ground
electrode.
[0135] Responses of the VEP were elicited by strobe flash test
stimuli generated by a GRASS PS-2 photostimulator directed toward
one eye with the other eye covered by a patch. Responses were
differentially amplified (GRASS P-15D preamp), displayed on a
TECTRONIX #564 oscilliscope and then averaged by a MACINTOSH IIx
computer using LABVIEW.
[0136] It was found that the reconstructed retina can produce a
light-evoked electrical response in the visual cortex whereas the
unreconstructed fellow eye showed little or no response to the same
light stimulus.
EXAMPLE 8
[0137] The procedures of Example 1 were repeated except as
noted.
[0138] With indications that neural activity is generated in the
central nervous system by the photoreceptor transplant and the
reconstructed retina the question arises at to whether this neural
activity can be processed appropriately by the central nervous
system to produce an appropriate behavioral response to the sensory
stimuli. Previous studies have shown that neural transplants to the
brain can restore appropriate behavioral activity (Bjorklund et
al., Neural Grafting in the Mammalian CNS. Elsevier, Amsterdam,
1985). Klassen and Lund Proc. Natl. Acad. Sci. USA 84: 6958-6960,
1987; and Exp. Neurol. 102: 102-108, 1988 have shown that neural
transplants can restore the pupillary reflex mediated by
intracranial transplantation of embryonic retinas thus showing that
neural transplants consisting of sensory tissue are capable of
mediating a behaviorally appropriate response to sensory
stimulation.
[0139] Rats with dystrophic retinas received a photoreceptor
transplant as described in Example 1. At various post-surgical time
intervals (E.G., 2, 4 and 8 weeks, etc.) animals were anesthesized
and held in a stereotaxic device. An infrared video camera was
focused on the eye through an operating microscope and the eye
illuminated with infrared light. To test for pupillary reflex, a
light beam controlled by a camera shutter within the operating
microscope was used. This light was focused on the eye. The
pupillary response to the light at graded intensities (intensity of
the light was controlled by neutral density filters) was recorded
by video camera connected to a frame grabber system. The pupillary
reflex was then analysed using automated imageprocessing software
(ULTIMATE, GTFS, Inc.)
[0140] It was found that retinas reconstructed with photoreceptor
transplants do in fact show a comparatively normal pupillary reflex
to light (pupillary constriction) whereas the fellow dystrophic eye
shows only a minimal reflex that is aberrant in form (pupillary
dilation). The results are shown in FIG. 28a, 28b, 28c and 28d.
Panels a and b are of the reconstructed retina. Panel a shows the
iris at light onset whereas panel b shows the same eye at 5 seconds
after light onset. Comparison of panel a to panel b shows a normal
pupillary constriction mediated by light. Panels c and d show the
fellow blinded eye that received sham surgery with panel c showing
the iris at light onset and panel d showing the iris 5 seconds
later. Comparison of panels c and d show an increase in pupil size
with light. This response is aberrant in form and is characteristic
of individuals suffering from severe retinal dystrophy of a
photoreceptor type.
[0141] These results show that neural transplantation can
reconstruct the host's own sensory end organ--in this case the
eye--to restore an appropriate behavioral response (i.e., the
pupillary reflex) to sensory stimuli. These results have profound
significance for the feasibility of the restoration of vision by
photoreceptor transplantation.
EXAMPLE 9
[0142] The procedures of Example 1 were repeated except as
noted.
[0143] Photoreceptors were taken from mature macaque retina
(animals sacrificed for other research) or the retina of donated
human eyes (obtained from the St. Louis Eye Bank) using vibratome
sectioning of the flat-mounted retina to isolate the intact outer
nuclear layer. Hosts were mature macaque monkeys treated with
iodoacetic acid (30 mg/kg given on 3 successive days) which
selectively eliminates host photoreceptors in non-macular areas of
the retina while leaving the remaining retina intact. This
treatment did not compromise central vision and therefore
maintained sight required for behavioral and physiologically
important functions (e.g., locating of food and water, visually
guided locomoter activities, grooming, maintenance of circadian
rhythms).
[0144] The isolated outer nuclear layer was transplanted following
a pars plana vitrectomy (a standard surgical technique) using a
trans-scleral approach to the subretinal space. The photoreceptors
were inserted under a focal retinal detachment induced by the
formation of a subretinal bleb. The bleb was created by the
infusion of ophthalmic balanced salt solution. The reconstructed
retina was reattached to the back of the eye by pneumatic tamponade
with the transplanted photoreceptors interposed between the retina
and the underlying pigment epithelium. Daily injections of
cyclosporin A and dexamethasone were made to suppress any possible
transplant rejection.
[0145] It was found human photoreceptors survive transplantation to
the non-human primate eye for as long as tested (2 weeks). These
results indicate that mature human photoreceptors can be
transplanted to the non-human primate eye. Since the non-human
primate eye is almost identical to the human eye it is expected
that human photoreceptors can be successfully transplanted to the
human eye.
[0146] From the foregoing description those skilled in the art will
appreciated that all aspects of the present invention are realized.
The present invention provides an improved surgical instrument that
is adapted to provide cell organization during transplantation of
the photoreceptors. With the surgical instrument of this invention
cell organization is maintained during photoreceptor, RPE, and
choroidal transplantation while minimizing trauma to the
transplanted tissues, the host eye and retina. It is believed that
retina reattachment and subsequent substantially normal function of
the reconstructed retina, in view of the transplant, is thereby
facilitated. The present invention provides an improved surgical
instrument that is constructed to allow relatively large expanses
of the RPE, choroidea, and photoreceptor cell matrix or column to
be transplanted to a sub-retinal space. Maintaining normal layer
configuration of the photoreceptors, RPE, and choroidea allows
these tissues to be transplanted to the appropriate position within
the eye. The subsequent integration of the transplanted
photoreceptors, RPE, and choroidea with the blinded retina
facilitates reconstruction of the blinded retina. The present
invention provides an improved surgical instrument that allows
appropriate retinotopic positioning. The present invention provides
an improved surgical instrument that protects photoreceptors from
damage as the surgical device is positioned in the eye. The present
invention provides a method of photoreceptor or retinal pigment
epithelium isolation and transplantation that, maintains to the
extent possible the normal organization of the outer nuclear layer
and these other tissues. The present invention provides a method of
cell and tissue isolation by which cells can be isolated without
disruption of their intercellular organization. With the method of
this invention retinal cells, such as retinal photoreceptors can be
isolated without the disruption of the intercellular organization
of the outer nuclear layer or other layer of the retina, RPE, and
choroidea.
[0147] A number of features of the transplanted cells are that they
are and remain alive; they produce opsin, important for
phototransduction; they are functional (i.e., activated by light);
and the transplanted photoreceptors activate a previously blinded
retina in a light dependent fashion.
[0148] Attachment of retinal tissue to the gelatin substrate allows
extended periods of in vitro culture of retinal tissues by:
maintaining organization of tissue in culture; and allowing for a
better viability of cultured tissue.
[0149] While a number of embodiments have been shown and described,
many variations are possible. Photoreceptors can be transplanted to
retina in which the host's or recipient's photoreceptors are lost
by environmental (constant light) or inherited defects. (See: S. E.
Hughes and M. S. Silverman (1988) in "Transplantation of retinal
photoreceptors to dystrophic retina", Soc. Neurosci. Abstr., 18:
1278.) Furthermore transplanted photoreceptor cells maintain basic
characteristics of normal photoreceptor cells by producing opsin
and maintaining an intercellular organization and apposition to the
host retina that is similar to that seen in the normal outer
nuclear layer. The surgical instruments may be larger for use in
humans. Other approaches to the subretinal space may be used, e.g.,
trans-scleral, choroidal. Other substrates besides gelatin can be
used, e.g. agar, agarose, in fact improved substrates could include
factors that can be integrated into gelatin, for example,
neurotrophic factors). It is believed that attachment to gelatin or
equivalent substrates will allow prolonged in vitro culture, or
cryogenic freezing, and similar storage, while allowing for the
maintenance of tissue organization and viability. Finally, it is
believed that other methods of attaching retina to substrate can be
used, such as lectins, or photo-activated cross-linking agents.
[0150] It has been shown that this invention provides a method to
isolate the intact photoreceptor layer. This is significant because
it will be necessary to maintain tight matrix organization if
coherent vision is to be restored to the retina comprised by the
loss of photoreceptors. A surgical approach has been disclosed
which minimizes trauma to the eye and allows controlled positioning
of sheets of transplanted photoreceptors to their homotopic
location within the eye. In addition these methods for
transplantation and isolation of photoreceptors could be utilized
to prepare and transplant other retinal layers so that selected
populations of retinal cells can be used in other neurobiological
investigations and clinical procedures. It is believed that these
other retinal layers, once they are flattened, appropriately
sectioned, and appropriately affixed to a stabilizing substrate or
base, could be prepared for transplantation, storage (e.g., in
vitro, cryogenic), or culturing similar to the methods described
herein for photoreceptor layers.
[0151] The necessity for prompt re-vascularization typically limits
the ability to transplant most neural tissue, but not
photoreceptors. The photoreceptor layer of a retina and the ("RPE")
is non-vascularized. Non-vascularized tissue shows the least amount
of tissue rejection. Consequently, it is believed that genetically
dissimilar photoreceptor cells may be transplanted in accordance
with the present invention. Matching of host and donor
histocompatibility antigens will probably be necessary for
transplantation of the retinal pigment epithelium and
choroidea.
[0152] Photoreceptors can be transplanted when developing or when
mature. Not only can mature rat photoreceptors be transplanted, but
mature photoreceptors from human donors can be transplanted as
well. This is significantly different from neurons which must be
immature in order to be transplanted. At present the reason for
this difference is not known but has obvious importance for retinal
and neural transplantation research in general.
[0153] Finally, transplanted photoreceptors activate the host's or
recipient's dystrophic retina in a light dependent manner that
closely resembles the activation pattern seen in normal retina.
[0154] In view of the above, it will be seen that the several
objects of the invention are achieved and other advantages
attained.
[0155] As various changes could be made in the above surgical
instruments, compositions of matter and methods. Without, departing
from the scope of the invention, it is intended that all matter
contained in the above description or shown in the accompanying
drawings shall be interpreted as illustrative and not in a limiting
sense.
* * * * *