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Gelatin is an example of a preferred support material which is flexible, lacks toxicity to neural tissue and has the ability to dissolve at body temperature.
Another alternative is to use biodegradable polymers such as ethylene-vinyl acetate copolymer Elvax 40W, DuPont Chemical Co. Polysciences, Inc. See, e. Data Sheet Advantageously, the gelatin or other support composition may additionally serve as a carrier for any of a number of trophic factors such as pharmacologic agents including immunosuppressants such as cyclosporin A, anti-inflammation agents such as dexamethasone, anti-angiogenic factors, anti-glial agents and anti-mitotic factors.
Upon dissolution of the support composition, the factor or agent becomes available to impart the desired effect upon the surrounding tissue. The dosage can be determined by established experimental techniques. With appropriate enzymatic digestion, using techniques disclosed by Pfeffer, B. Such a freshly harvested intact monolayer of RPE cells may be immediately apposed as an intact monolayer to a support, such as a thin collagen sheet, supplemented with an overlayer or substrate of gelatin, allowing several hours for adhesion prior to transplantation.
This procedure may be useful for transplantation of RPE cells obtained in a biopsy, provided sufficient RPE tissue is obtained from the biopsy Mammals - Lucifer Wong - Mammals (Vinyl) an intact monolayer. In most cases, however, it is preferable to culture the harvested RPE cells on an attachment substrate so that a monolayer of RPE cells may be prepared.
Culturing the RPE cells before harvesting is also preferred both to allow for the production of larger populations of RPE cells from a small amount of harvested tissue and to allow for a period of observation to ensure that the RPE cells to be transplanted are healthy and functional. For proper culturing, harvested RPE cells contained are apposed to a substrate to which they will attach and grow, and which is capable of being maintained in culture conditions appropriate for efficient growth of RPE cells.
Apposition of RPE cells may be accomplished by pipetting a solution containing a population of RPE cells onto the substrate. Intact sheets of harvested RPE cells in a physiological solution may also be physically Mammals - Lucifer Wong - Mammals (Vinyl) via a wide-bore pipette onto the substrate and manipulated with a fine camel-hair brush if necessary.
Attachment substrates which are suitable for growth of RPE cell cultures include plastic culture ware or glass culture chamber slides coated with collagen for cell attachmant. Culturing harvested RPE cells can be accomplished by any suitable method for obtaining a confluent monolayer of RPE cells. Appropriate culturing techniques are described in Pfeffer, B. An implant laminate 1 is formed as depicted in FIGS.
Thus, a monolayer of RPE cells 5 is first cultured on a suitable substrate for growth. RPE cells will grow on plastic culture ware A. However, to aid in removal of the monolayer of RPE cells 5 from the culture ware A, the culture ware A may be coated with a substrate 9 for the RPE cells, such as agarose or fibrin.
See FIG. The substrate 9 is in turn coated with a thin microns layer of collagen 7, to which the monolayer of RPE cells 5 readily attach, and which will serve as an anchor for the RPE cells in the post-transplantation period.
If agarose is used as the substrate 9, the agarose is activated so that the collagen 7 will adhere to it by treating the surface of the agarose with an activator such as cyanogen bromide, as disclosed by Axen, R.
The implant laminate 1 comprising a monolayer of RPE cells 5, attached to the collagen anchor 7 and an overlayer of gelatin 3, may then be physically removed from the culture ware A by slicing between the culture ware A surface and the collagen 7 with a razor C, as depicted in FIG. The implant laminate 1 may also be removed from the culture ware and substrate 9 enzymatically, as depicted in FIG. The underlying substrate 9 may be removed from the implant laminate 1 by application of an enzyme specific to the material comprising the substrate 9.
If the substrate is agarose, agarase, an enzyme specific to agarose, may be used to dissipate the agarose. To aid in dissipation of the substrate 9, the culture ware A may comprise a cell culture insert membrane, containing an inner membrane A-1 with a porous bottom, and an outer, solid membrane A The porous e. These pores facilitate penetration of the solution B containing the enzyme into the substrate 9 so that it may be broken apart, and the implant laminate 1 removed from the culture ware A.
As depicted in FIG. The implant 1a is transplanted to the subretinal area at the posterior pole 76 of the host eye after detachment of the retina, as portrayed in FIG.
The gelatin overlayer dissolves within hours after insertion of the implant 1a into the host eye. After transplantation, the retina reattaches, and the monolayer of RPE cells, anchored to the layer of collagen, is sandwiched between the choroid and the retina. To transplant the implant, the host eye is prepared so as to reduce bleeding and surgical trauma.
The preferred Mammals - Lucifer Wong - Mammals (Vinyl) approach in the human, FIG. The instrument 20 is advanced through the sclera and choroid 78 and to the ora serrata 74 as illustrated in FIG. The instrument detaches the retina as it is advanced under the retina and into the sub-retinal space to the posterior pole 76 of the eye. Preferably, an instrument comprising an elongate tube having a flat, wide cross-section may be used so that the implant may be drawn into the elongate tube for protection as it is transported through an appropriate sized incision in the sclera or choroid.
Advantageously, the fluid may additionally contain anti-oxidants, anti-inflammation agents, anesthetics or agents that slow the metabolic demand of the host retina.
If the instrument does not include a lumen, the retina is detached by subretinal irrigation or 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 implant is then transplanted by retracting the tube containing the implant from the eye while simultaneously gently ejecting the implant from the tube. The instrument is then carefully withdrawn out of the eye. Retinal reattachment occurs rapidly and the monolayer of RPE cells is held in place in a sandwich-like arrangement between Mammals - Lucifer Wong - Mammals (Vinyl) retina and the choroid.
The incision may require suturing. Except for the point of entry, the surgical technique is essentially the same as outlined for the transscleral or choroidal approaches.
A transverse incision 70 is made in a cornea 72 and the instrument 20 containing the implant is advanced under the iris, through the cornea 72 and to the ora serrata 74 as illustrated in FIG. The iris should be dilated for example, with topical atropine. The edges of the corneal incision are abutted and sutured if necessary to allow healing. The transcorneal approach is preferred for rodents because it has been found to reduce bleeding and surgical trauma.
Nevertheless, a transscleral or choroidal approach is preferred for humans to avoid scarring of the cornea which may interfere with visual acuity. A further surgical approach is to diathermize in the pars planna region to eliminate bleeding. The sclera is then incised and the choroidal and any native epithelial tissue is diathermized. The surgical tool is then inserted through the incision, the retina is intercepted at the ora serrata and the implant is Mammals - Lucifer Wong - Mammals (Vinyl) in the subretinal area otherwise as outlined elsewhere herein.
In yet a further surgical approach, entry is gained through the pars planna 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. As discussed previously in connection with the make up of the support compositions used in accordance with the invention, after transplantation, the implant is exposed to a predetermined set of conditions, such as exposure to heat or bodily fluids.
Upon the appropriate amount of exposure, the support composition is dissipated or will not otherwise impede normal eye tissue function of the host eye and the transplanted population of RPE cells. RPE cells were taken from the sub-retinal area of donated human eyes obtained from the Missouri Lions and St. Louis Eye Banks following corneal removal.
Immediately after corneal removal, the eye was pinned by the optic nerve stump into an eye cage and placed into an eye jar. The eye was processed in a sterile environment. After loose connective tissue and muscle were carefully trimmed from the eye, it was placed upright on a sterile plate and the anterior segment with the adherent vitreous was lifted out of the eye cup.
Four slits were cut radially toward the optic disc with the eye lying flat in a petri dish. The neural retina was then separated at the optic disc and removed. The cells were released from Bruch's membrane by gently brushing the surface of the RPE cells with the polished tip of a pasteur pipette and pipetting the culture medium in the shell. Contrassegna il permalink. Rispondi Cancella risposta Scrivi qui il tuo commento Inserisci i tuoi dati qui sotto o clicca su un'icona per effettuare l'accesso:.
Nome obbligatorio. Only then can I switch and start a new one. Hair, cilia - natural? Hair I use special doll, and also from a natural material of a goat, a llama, a sheep Cilia are natural squirrels. I paint the doll with dry decorative cosmetics and oil. That it harmoniously, beautifully and correctly looked, it is necessary to know anatomy, sculptural modeling, to understand hairdressing business, be able to sew clothes, make shoes, know the basics of make-up and much more.
Moreover, one should burn with the desire to create dolls and enjoy tremendous perseverance. Basically, the work is bought by collectors, among them there are those who follow my work and ask to show everything I do.
The creation of the image is connected with the author's state of mind. This is a complex process. Each doll is done by hand, so it turns out with its character, look. I still have a hard time modeling the face and hands. I'm still learning a lot, I'm doing a lot of things. Finished plastic parts are baked in the oven at a certain temperature. If something went wrong, I want to start again. I do not presume to make dolls under the order. Duplicate - it's not interesting, I think it's better to create a new one.
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