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Method Article

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

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DOI:

10.3791/56469

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February 12th, 2018

 ,  ,  ,  , 
1Low Vision Research Centre of Milan, 2Department of Ophthalmology, A. Fiorini Hospital, Sapienza University of Rome, 3Glaucoma and Low Vision Study Center, Department of General Surgery and Organ Transplants, University of Bologna, 4Department of Sense Organs, Faculty of Medicine and Odontology, Sapienza University of Rome

In This Article

Summary

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

Abstract

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Introduction

Cell therapy, consisting of the systemic or local injection of stem/progenitor cells in the injured area to treat multiple chronic disorders, has drawn close attention in the last decade1. Since the 1990s, growth factors (GFs) have been studied for their potentially therapeutic role in retinal atrophy2. In fact, many human cells can produce GFs, which are specific proteins that are able to block or slow down apoptosis, i.e., the programmed death of cells3.

It is known that dry age-related macular degeneration (AMD) is an atrophic retinal disease where gradual and irreversible cell death involves injury to the photoreceptor layer and, consequently, the loss of central visual function4. AMD is the leading cause of blindness in people over 55 years of age in developed countries and accounts for 80% of all macular degenerations, which lack an effective treatment to date.

Several studies have shown that there are various sources from which autologous GFs can be obtained. These comprise different cell types, including adipose stromal cells derived from orbital fat, platelets derived from platelet-rich plasma (PRP), and adipose-derived stem cells (ADSCs) included in the stromal vascular fraction (SVF) of adipose tissue5,6,7. The current GF set ensures retinal neuroenhancement, and research conducted by Filatov, Meduri, Pelaez, and Limoli has demonstrated that autologous fat transplantation (AFT) is effective8,9,10.

Moreover, a prior study showed significant improvements in electroretinogram (ERG) data, recorded post suprachoroidal autologous graft, in dry AMD-affected eyes11. The surgically grafted tissue in the suprachoroidal space modulated the paracrine secretion of retinal cells, delaying their apoptosis6,7,12. Considering outer nuclear layer thickness, the histological examination of the retina of guinea pigs has shown that GFs could have a trophic effect on the retina. Therefore, the direct or indirect use of GFs can potentially bring therapeutic benefits through a balanced relation between molecular inducers and inhibitors6,7,12.

The purpose of this method is to assess whether the suprachoroidal graft of adipocytes, ADSCs in SVF and PRP can improve best corrected visual acuity (BCVA) and microperimetry (MY) responses in dry AMD-affected eyes. This study aims to demonstrate the therapeutic effect of autograft on the basis of its GF production, according to the cited literature6,7,12,13.

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Protocol

The study protocol was approved by the Ethics Committee of the Low Vision Academy and all subjects signed a written consent in accordance with the Helsinki Declaration. This research study has received ethical approval from both Loughborough and Sheffield Universities.

NOTE: The inclusion and exclusion criteria of dry age-related macular degeneration patients to receive suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) is described in Table 1.

1. Diagnosis of Dry Age-related Macular Degeneration Patients

  1. Ascertain the diagnosis with confocal scanning laser ophthalmoscope, SD-OCT, and MY.
  2. Evaluate each group's BCVA for far and near distance. Measure VA for near vision (close-up) in points (Pts). Measure BCVA at time 0 (T0), 90 (T90), and 180 days (T180) compared to early treatment diabetic retinopathy study (EDTRS) charts at 4 meters in logMAR.
  3. Record electrical scotopic, mesopic, and photopic cell activity, or flash ERG, according to the standards set in 2009 by the International Society for Clinical Electrophysiology of Vision (ISCEV)11.

2. Anesthetization

NOTE: The gold standard in anesthesia during LRRT is topical anesthesia, reinforced by sub-tenon's infiltration of anesthetic and sedation. In specific cases, general anesthesia is preferred.

  1. Obtain corneal and conjunctival anesthesia by applying topical local anesthetics instilled dropwise 15 - 20 min before the surgery with lidocaine at 4% and ropivacaine at 1%.
  2. Inject anesthesia by infiltration directly into sub-conjunctival and subtenon's spaces.
  3. Use local infiltration both in the abdominal region, before the adipose tissue is extracted, and in the sub-conjunctival and sub-tenon's spaces, 12 mm from the limbus. Adopt local anesthetic of carbocaine or marcain mixed with 1,200 IU epinephrine.
  4. Provide intraoperative sedation through the anesthetic, which can be performed properly by using fentanyl as a narcotic analgesic through repeated small boli. The dosage is generally 0.025 mg of fentanyl with 1 mg of midazolam per bolus.

3. Limoli Retinal Restoration Technique Preparation

NOTE: This technique represents a variant of Pelaez's intervention by which orbital autologous fat is transplanted in the subscleral space1,6,7,12. Surgically grafted cells can produce many GFs with neurotrophic and angiotrophic properties in the surrounding tissue, choroid, and retina18,19,20,21,22,23,24,25. In LRRT, the distance between grafted autologous cells and choroid is reduced by means of deep sclerectomy, and the contact area between the stalk and choroid is expanded to promote the paracrine autologous cell secretion into the choroidal flow9,10,14.

  1. Perform proper disinfection of each eye before surgery with cellular grafting between the choroid and sclera, a procedure called Limoli Retinal Restoration Technique (LRRT)15,16,17.
  2. Graft the ADSCs, obtained by Coleman et al. and Lawrence's technique (Figure 1) from abdominal fat, in the SVF in the sovrachoroidal space15,16,17.
  3. Infiltrate adipose pedicle with platelets derived from PRP gel obtained through the following steps.
  4. Centrifuge blood6,12 and collect platelet-rich plasma (PRP). The stimulus to platelet degranulation causes GF release in the adipose pedicle6,12.

4. Technical Specifications and Strategy

Note: Fat tissue is collected and purified from the abdominal subcutaneous layer of patients, according to the Lawrence and Coleman technique17(Table of Materials).

  1. Manually harvest 10 mL of fat tissue from the abdominal subcutaneous layer of each patient, using a 3-mm blunt cannula connected to a locking syringe, according to the Lawrence and Coleman technique17 (Figures 2A/2B).
  2. Separate pure SVF of fat tissue from blood, fat, oil, and liquid by centrifugation for 5 min at 1,500 x g at 20 °C (Figure 2C). The SVF is very rich of ADSCs17.
  3. Collect 8 mL of human peripheral blood with a 22 G needle and in a separate tube for PRP preparation.
  4. Centrifuge the collected blood for 5 min at 1,500 x g at 20 °C (Figure 2D). In LRRT, the ensuing changes result in better survival of the autologous fat graft, ADSC proliferation, which favors increased choroidal perfusion, and a more comprehensive modulation of the action of those factors that are secreted only by fat7,11,17.
  5. Build the suprachoroidal pocket (more details in step 4, in particular 4.4 and 4.5) to accommodate the graft obtained from orbital fat and saturate the residual volume of this pocket with a mixture of ADSCs from SVF and PRP, obtained according to the Lawrence and Coleman technique17.

5. Suprachoroidal Autograft by LRRT (Limoli Retinal Restoration Technique): Surgical Procedure and Technical Details

  1. Anchor the sclera with 6-0 silk suture, near the inferior-temporal limbus.
  2. Open the subconjunctival and subtenonian space at 11 mm from the inferior-temporal limbus, using 5.5" Westcott Tenetomy curved scissors.
  3. Insert the Limoli-Basile conjunctival retractor in this space to make a scleral surgical field.
  4. Using a 5-mm crescent knife angled bevel up, pre-cut a flap on the side in the sclera at 8 mm, from the limbus. The flap hinge is always radial and to the left of the surgeon.
  5. In the inferior-temporal quadrant, at 8 mm from the limbus, open a deep scleral door of about 5 mm on the side by radial hinge by using a crescent knife, angled bevel up. Carry out sclerectomy at an adequate depth to view the slate color of the choroid.
  6. Create a gap by removing a little operculum in the distal part of the flap, in order to facilitate blood circulation in the subsequent suprachoroidal autograft.
  7. Extract with ophthalmological forceps the orbital fat from a gap above the inferior oblique muscle. Make sure the extracted fat is sufficiently vascularized to allow it to survive after its implantation.
  8. Gently place the autologous fat flap on the choroidal bed and suture with choroidal 6/0 polyglactin fiber at the proximal edge of the door.
  9. Suture the scleral flap to avoid compression on the fat pedicle or on its nutrient vessels.
  10. Infiltrate the stroma of the fat pedicle with 1 mL of PRP gel (obtained by centrifugation of the blood material, separation of the component, and platelet degranulation26) using a 30 G angled (30°) cannula.
  11. Prepare the sides of the conjunctiva for the suture. Then, remove the conjunctival retractor.
  12. Suture the conjunctiva, using 6/0 polyglactin fiber.
  13. Before closing, leave a space to insert into the subscleral space, between the flap, the choroid, and the choroidal autograft, a small flexible plastic tube with the autologous fat graft.
  14. Saturate the residual space between the autologous fat graft, choroid, and scleral flaps with 0.5 cc of SVF (rich of ADSCs), previously prepared in step 3.2, by a small flexible plastic tube, inserted into the scleral pocket.
  15. After saturating the residual space, close the suture.
  16. After surgery, administer three days of antibiotic therapy with 500 mg azithromycin. Also, provide eye-drop therapy with an antibiotic and steroid combination, such as Chloramphenicol and Betamethasone, for about 15 - 20 days.
    NOTE: An autograft made up of fat cells, ADSCs from SVF, and PRP has now been obtained26. Reduce the distance between the grafted autologous cells and choroid by deep sclerectomy to stimulate paracrine secretion of autologous cells into the choroidal flow. For the same purpose, expand the area of contact between the stalk and choroid.

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Results

Using the procedure presented here, two groups of dry AMD-affected patients, with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR), were enrolled in the study. Group A, including 11 eyes of 11 patients, received suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT), whereas group B, including 14 eyes of 14 patients, was used as a control group.

Student's t-test and th...

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Discussion

The primary purpose of this study was to evaluate whether the suprachoroidal graft of adipocytes, ADSCs in SVF, and PRP could improve VA and retinal sensitivity in dry AMD-affected eyes over time. Another main objective was to demonstrate possible therapeutic effects of these cells, based on the recent literature, since several preclinical studies have suggested that GF-based therapy could be useful for patient care in several diseases.

In fact, some studies have shown that autologous human in...

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Disclosures

Presented at ARVO 2015, May 3-7 - Denver, CO - USA.

Acknowledgements

The authors have no acknowledgements.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Blunt cannula, 3 mm. Mentor, Santa Barbara, CA.
Luer-LokTM syringe. BD Biosciences, Franklin Lakes, NJ.
Regen-BCT tube. RegenKit; RegenLab, Le Mont-sur-Lausanne, CH.
Centrifuge RegenPRP Centri. RegenLab, Le Mont-sur-Lausanne, CH.
BD Venflon Pro Safety 22G x 1.00 inch (0.9 mm x 25 mm). BD Biosciences, Franklin Lakes, NJ.
SPSS Statistics Version 19.0IBM Corp., Armonk, NY, USA.
Confocal scanning laser ophthalmoscope Nidek Inc, Fremont, CANidek F10 
Cirrus 5000 Spectral Domain-Optical Coherence TomographyCarl Zeiss Meditec AG, Jena, Germany SD-OCT 
Maia 100809 Microperimetry CenterVue S.p.A., Padua, Italy
Ocular electrophysiology electromedical system,C.S.O., S.r.l., Scandicci, Italy Retimax for ERG 

References

  1. Daftarian, N., Kiani, S., Zahabi, A. Regenerative therapy for retinal disorders. J. Ophthalmic Vis. Res. 5, 250-264 (2010).
  2. Thanos, C., Emerich, D. Delivery of neurotrophic factors and therapeutic proteins for retinal diseases. Expert. Opin. Biol. Ther. 5, 1443-1452 (2005).
  3. Cao, W., et al. In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Inv. Ophthalmol. Vis. Sci. 42, 1646-1652 (2001).
  4. Bhutto, I., Lutty, G. Understanding age-related macular degeneration (AMD): Relationships between the photoreceptor/retinal pigment epithelium/Bruch's membrane/choriocapillaris complex. Mol. Aspects Med. 33 (4), 295-317 (2012).
  5. McHarg, S., Brace, N., Bishop, P. N., Clark, S. J. Enrichment of Bruch's membrane from human donor eyes. J. Vis. Exp. (105), (2015).
  6. Kevy, S. V., et al. Preparation of growth factor enriched autologous platelet gel. Transactions of the Society for Biomaterials 27th Annual Meeting. , St. Paul, Minnesota, USA. April 24-29 (2001).
  7. Schaffler, A., Buchler, C. Concise review: adipose tissue-derived stromal cells-basic and clinical implications for novel cell-based therapies. Stem Cells. 25, 818-882 (2007).
  8. Filatov, V. P. Tissue therapy. Med. Gen. Fr. 11, 3-5 (1951).
  9. Pelaez, O. Retinitis pigmentosa. Cuban experience. , Editorial Cientifico Técnica. La Habana, Cuba. (1997).
  10. Meduri, R., et al. Effect of basic fibroblast growth factor on the retinal degeneration of B6(A)- Rperd12/J (retinitis pigmentosa) mouse: a morphologic and ultrastructure study. ARVO 2007 Annual Meeting. , Fort Lauderdale. May 6-10 (2007).
  11. Limoli, P. G., Vingolo, E. M., Morales, M. U., Nebbioso, M., Limoli, C. Preliminary Study on Electrophysiological Changes After Cellular Autograft in Age-Related Macular Degeneration. Medicine. 93 (29), 355(2014).
  12. Tischler, M. Platelet rich plasma: The use of autologous growth factors to enhance bone and soft tissue grafts. N. Y. State Dent. J. 68, 22(2002).
  13. Zuk, P. A., et al. Human adipose tissue is a source of multipotent stem cells. Mol. Biol. Cell. 13 (12), 4279-4295 (2002).
  14. Lin, K. J., et al. Topical administration of orbital fat-derived stem cells promotes corneal tissue regeneration. Stem Cell Res. Ther. 4 (3), 72(2013).
  15. Limoli, P. The retinal cell-neurorigeneration. Principles, applications and perspectives. The growth factors. , FGE Reg. Canelli (AT). 159-206 (2014).
  16. Coleman, W. P., et al. Guidelines of care for liposuction. J. Am. Acad. Dermatol. 45, 438-447 (2001).
  17. Lawrence, N., Coleman, W. P. Liposuction. J. Am. Acad. Dermatol. 47, 105-108 (2002).
  18. Kamao, H., et al. Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. Stem Cell Reports. 23 (2), 205-218 (2014).
  19. Dang, Y., Zhang, C., Zhu, Y. Stem cell therapies for age-related macular degeneration: the past, present, and future. Clin. Interv. Aging. 10, 255-264 (2015).
  20. Nebbioso, M., Livani, M. L., Steigerwalt, R. D., Panetta, V., Rispoli, E. Retina in rheumatic diseases: Standard full field and multifocal electroretinography in hydroxychloroquine. Clin. Exp. Optom. 94 (3), 276-283 (2011).
  21. Wang, P., Mariman, E., Renes, J., Keijer, J. The secretory function of adipocytes in the physiology of white adipose tissue. J. Cell. Physiol. 216, 3-13 (2008).
  22. Chen, G., et al. VEGF-Mediated Proliferation of Human Adipose Tissue-Derived Stem Cells. PloS One. 8, 73673(2013).
  23. Bagchi, M., et al. Vascular endothelial growth factor is important for brown adipose tissue development and maintenance. FASEB J. 27, 3257-3271 (2013).
  24. Carron, J. A., et al. Cultured human retinal pigment epithelial cells differentially express thrombospondin-1, -2, -3,and -4. Int. J. Biochem. Cell. Biol. 32, 1137-1142 (2000).
  25. Kim, S. Y., et al. Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid. Exp. Eye Res. 77, 433-445 (2003).
  26. Limoli, P. G., Limoli, C., Vingolo, E. M., Scalinci, S. Z., Nebbioso, M. Cell surgery and growth factors in dry age-related macular degeneration: visual prognosis and morphological study. Oncotarget. 7 (30), 46913-46923 (2016).
  27. Ueki, Y., Reh, T. A. EGF stimulates Müller glial proliferation via a BMP-dependent mechanism. Glia. 61, 778-789 (2013).
  28. Kozlowski, M. R. RPE cell senescence: A key contributor to age-related macular degeneration. Med. Hypotheses. 78, 505-510 (2012).
  29. Schneider, A., et al. The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis. J. Clin. Invest. 115, 2083-2098 (2015).
  30. Yin, Y., et al. Oncomodulin is a macrophage-derived signal for axon regeneration in retinal ganglion cells. Nat. Neurosci. 9, 843-852 (2006).

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Suprachoroidal GraftAutologous CellsDry AMDLimoli Retinal Restoration TechniqueStromal Vascular FractionPlatelet-Rich PlasmaAdipose-Derived Stem CellsConfocal Scanning Laser OphthalmoscopeSpectral Domain-OCTMicroperimetry Test
Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report
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