CuZn superoxide dismutase transgenic retinal transplants

Graefe's Arch Clin Exp Ophthalmol (1999)  Springer-Verlag 1999 237:336±341

Thomas Grasbon Eva Maria Grasbon-Frodl Bengt Juliusson Charles Epstein Patrik Brundin Anselm Kampik Berndt Ehinger

Received: 9 May 1997 Revised version received: 13 May 1998 Accepted: 17 June 1998 T. Grasbon ´ B. Juliusson ´ B. Ehinger Department of Ophthalmology, University of Lund, University Hospital of Lund, S-22185 Lund, Sweden

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T. Grasbon ( ) ´ A. Kampik University Eye Hospital, Ludwig-Maximilians-University, Mathildenstrasse 8, D-80336 Munich, Germany e-mail: Thomas.Grasbon @ak-i.med.uni-muenchen.de; Tel. +49-89-51603811, Fax: +49-89-51605160 E.M. Grasbon-Frodl Department of Neuropathology, Ludwig-Maximilians-University, Thalkirchner Str. 36, D-80337 Munich, Germany E.M. Grasbon-Frodl ´ P. Brundin Department of Physiology and Neuroscience, University of Lund, Sölvegatan 17, S-22362 Lund, Sweden C. Epstein Department of Pediatrics, University of California, San Francisco, California, USA

LABORATORY INVESTIGATION

CuZn superoxide dismutase transgenic retinal transplants

Abstract l Background: The morphology of retinal transplants is believed to depend on the extent of mechanical disruption of the donor tissue during the surgical procedure and on local factors of the host environment. We hypothesized that oxidative stress during donor tissue preparation and implantation further affects transplant development and investigated the effects of CuZn superoxide dismutase (SOD) overexpression on the survival and morphological development of mouse embryonic retinal transplants. l Methods: Retinae and livers from embryonic day 14±15 SOD overexpressing transgenic mice and CBA control mice were harvested under sterile conditions. In order to identify transgenic mouse embryos, the embryonic livers were analyzed via nondenaturing gel-electrophoresis for the presence of the human SOD protein. Neural retinae were transplanted as fragmented tissue into the subretinal space of albino BALB/c mice. At 4±8 weeks following transplantation,

Introduction Retinal transplantation is an experimental therapeutic strategy for degenerative diseases of the retina. In order to afford functional repair, retinal transplants need to be morphologically integrated into the host retina or to establish appropriate connections with target structures of the host brain. However, retinal transplantation to intraocular sites currently faces two major problems, namely dis-

the grafted eyes were fixed in Bouin's solution and processed for histological analysis. l Results: Both SOD transgenic and control retinal transplants had developed all retinal layers except for a ganglion cell layer and exhibited a similar extent of rosette formation. Computer-assisted, quantitative assessment of retinal graft volumes revealed a significant, around 58% increase in size of SOD transgenic transplants compared with controls. l Conclusions: Enhanced intracellular SOD levels do not seem to influence retinal transplant morphology as detected by light microscopy. However, volumes of the SOD trangenic transplants were found to be increased compared to control grafts.

turbed graft morphology, in particular rosette formation, and reduced ganglion cell survival. It has been suggested that traumatic disruption of the embryonic neuroretina plays a major role in rosette formation. In support of this view, it has been shown that fewer and larger rosettes are obtained when the transplantation technique is modified to a less traumatic handling of the donor retina which leaves larger tissue fragments intact [13, 31]. Furthermore, the embryonic neuroretina is exposed to hypoxia during dissection and transplantation.

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On a cellular level, tissue trauma and ischemia lead to membrane damage, edema, reduced nutritional support and energy impairment. Eventually, these pathophysiological changes will result in an increased generation of reactive oxygen species [7]. Since ganglion cells are among the first cells to differentiate in the developing retinal neuropeithelium [38], they might be particularly susceptible to trauma and the resulting oxidative stress. This would imply that most of the ganglion cells are irreversibly damaged already during the transplantation procedure. In the present study, we investigated the survival and development of retinal transplants taken from transgenic mice which overexpress the human cytosolic form of superoxide dismutase (CuZnSOD). SOD is the sole enzyme to detoxify superoxide radicals (O2 ´-) by reducing them to hydrogen peroxide (H2O2 ) and has been attributed a key role in preventing cell death. We therefore hypothesize that increased SOD levels might reduce oxidative stress during the transplantation procedure and thereby ameliorate ganglion cell survival and retinal transplant development.

Materials and methods Qualitative protein electrophoresis for specific SOD detection In order to obtain SOD transgenic mouse embryos, heterozygous male transgenic mice of strain TgHS 218/3, backcrossed for two generations to C57BL/6 mice, were mated with non-transgenic CBA females. Embryos (embryonic day 14±15, crown-rump length 11± 12 mm) were removed, the neural retinae and livers dissected. The embryos carrying the transgene were identified by qualitative demonstration of human CuZnSOD in the liver, using nondenaturing gel electrophoresis [8]. Briefly, the individual livers were homogenized in 1 mM EDTA and 0.5% NP-40, and 10±20 ml of each supernatant was loaded onto a nondenaturing polyacrylamide gel. After electrophoresis, the CuZnSOD bands were visualized with an activity stain containing riboflavin and nitroblue tetrazolium. The presence of the transgene was confirmed by the presence of a human CuZnSOD band and by the formation of human-mouse heterodimers. Extensive biochemical characterization of the strain of CuZnSOD transgenic mice used in the present study has previously demonstrated around 3 times higher levels of CuZnSOD in the CNS than in control mice [8, 23]. Retinal transplantation technique Retinae from SOD transgenic and control embryos were separately stored until transplantation (approximately 1±5 h) at 4 C in a chemically defined medium [16, 30]. The retinae were transplanted as

Table 1 Basic parameters of retinal transplants of SOD trangenic and control mice. In both groups, subretinally located transplants showed complete morphological differentiation, whereas epiretinally or vitreally located grafts matured only incompletely

fragmented tissue into the subretinal space of adult albino BALB/ c mice. The number of donor retinae that had been successfully implanted was documented for each graft recipient (Table 1). The graft recipients were deeply anesthetized with xylazine (Rompun; 10 mg/ kg body weight i.p.) and ketamine (Ketalar; 80 mg/kg i.p.). Each donor retina was sucked up into a thin plastic tube which was connected to a 10-l Hamilton microsyringe. The tube was introduced to the subretinal space through a small scleral incision at the equator of the right eye, and retinal tissue was injected into the subretinal space. The animals were treated in accordance with the Association for Research in Vision and Ophthalmology (ARVO) resolution on the use of animals in research as well as institutional guidelines. Histology At 31 or 57 days following transplantation, mice were sacrificed by CO2 asphyxia and cervical dislocation on dry ice within around 40± 60 s, followed by enucleation. Approximately 2 min later, the eyes were fixed in Bouin's solution for 5 h, intensively rinsed in 0.1 M Sùrensen's phosphate buffer (SPB) and dehydrated in sucrose concentrations increasing up to 20%/SPB. Prior to cryostat sectioning, the eyes were embedded in 30% egg albumin and 3% gelatin in H2O. Serial 12-m-thick sections were mounted directly on gelatin-coated slides and stored at Ÿ20 C. Every third section of all transplants was stained for hematoxylin and eosin (H&E). One representative H&E-stained section of four randomly chosen transplants of each group was selected for cell counting which was performed in blind manner. Cell nuclear density was calculated deviding the number of counted nuclei by the measured transplant area of the respective section. Representative sections of each transplant were labeled with an antibody against human Protein Gene Product 9.5 (PGP 9.5, dilution 1:3000; Ultraclone, Isle of Wight, UK), which is reported to be specific for retinal ganglion and horizontal cells in a variety of mammals including mice [2]. For immunocytochemistry, unspecific peroxidase was blocked in 3% H2O2 in phosphate-buffered saline for 10 min, followed by 30 min preincubation in 2% normal horse serum (Dakopatts, Glostrup, Denmark). After incubation in primary antiserum at 4 C for 3 days, the sections were incubated with the appropriate biotinylated secondary antibody (1:200; Dakopatts) and an avidin-biotin-peroxidase kit (Vector, Burlingame, Calif., USA). Finally, sections were developed in 3,3©-diaminobenzidine solution (DAB-Kit, Vector) for about 2 min, rinsed in distilled water, dehydrated in increasing alcohol concentrations and coverslipped using Entellan (Merck) as mounting medium. Computer-assisted image analysis Computer-assisted image analysis was performed by encircling the outlines of the grafts on every third section through each transplant, using the Neotech Image Grabber (version 2.1) and NIH-Image (version 1.52) software. Final graft volumes were calculated taking into account the total graft area measured per transplant, section thickness (12 m) and sampling frequency (1:3).

Number of transplants Average number of donor retinae per transplant Subretinal location Fully matured grafts 31 days survival 57 days survival Mean graft volume (l) per donor retina

SOD transgenic group

Control group

7 1 5 5 7 0 0.098

7 1.2 4 4 4 3 0.062

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Results Transplant survival and location In 14 of 33 graft recipients (42%), surviving grafts were histologically verified in both epiretinal or vitreal and subretinal location. With regard to overall graft survival there was no major difference between the SOD transgenic transplants and controls. The basic parameters of the 14 transplants are summarized in Table 1. Transplant morphology Underneath the transplants, the pigment epithelium of the graft recipients was usually well preserved (Fig. 1). As revealed by light microscopy, the SOD transgenic and control retinal transplants showed similar morphological features. Except for a ganglion cell layer, all retinal layers were identified and were typically arranged in rosettes (Fig. 1). An outer limiting membrane was regularly seen Fig. 1 Representative photomicrographs showing retinal transplants (T) of the SOD transgenic (A) and control (B) groups. Note the subretinal location of the transplants between the host's neuroretina (H) and retinal pigment epithelium (arrow). Numerous rosettes (asterisks) consisting of an inner and outer nuclear layer as well as an inner and outer plexiform layer were observed in transplants from both groups. H&E; bar 100 mm

whenever the sections went through the center of a rosette. Photoreceptors in both groups formed rod and cone inner segments as well as long outer segments. All transplants were highly vascularized. In one case, an anastomosis to the host retinal blood vessels was observed. In both groups, immunocytochemistry for PGP 9.5 revealed few immunoreactive cells, mainly located at the outer border of the inner nuclear layer or at the graft-host border (Fig. 2). These cells characteristically exhibited long, horizontally arborized processes and a rather small soma diameter and were interpreted as horizontal cells. No observable differences in the number of PGP-positive cells were seen between the two groups. Quantitative assessment of graft volumes Rodent retinal transplants performed using a technique similar to ours have been shown to mature according to the normal developmental timetable [1]. Taking into consideration the growth characteristics of the developing rodent retina [3, 34], all transplants analyzed in the present

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Fig. 2 Photomicrographs of SOD transgenic (A) and control (B) retinal transplants (T) immunostained for PGP 9.5. Immunoreactive, presumably horizontal cells were mainly located in regions corresponding to the inner nuclear layer. H Host retina. Bar 50 mm

SEM=0.115 l0.013, n=5; control group: meanSEM=0.068 l0.009, n=4; 69% difference; unpaired Student's t-test, P