Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

2.2. Preparation of the RPE Cell Sheet from PIPAAm-Grafted Plates

RPE cultured either on PIPAAm-grafted or non-grafted plates was started with same cell concentration (3 ¥ 105 cells/ml) in 35 mm culture plates. Changing the temperature of the culture plate from 37° C to 20° C, we could collect the cultured RPE as cell sheet as reported previously.5

2.3. Quantification of Collagen Type IV and BDNF Expression

The amount of collagen type IV was determined by enzyme-linked imunosorbent assay (ELISA). The amount of BDNF (Promega Co.) protein were also quantified according to the manufacture’s instruction.

2.4. Western Blot Analysis

Western blot analysis was performed using anti-chondroitin sulfate A (Seikagaku corporation, Tokyo, Japan) as we reported previously.6

2.5. Apoptosis of RPE and Flowcytometry

When RPE cultured on PIPAAm plate was detached from the plates, RPE cultured on normal culture plates was collected by 0.25% trypsin. These collected cells were cultured in the same medium coated with 1.5% agarose culture plate, which unable the cells to attach the culture plates, as reported previously.7 Rat RPE collected as cell sheet and cultured with unattached condition for 3 hr was recovered. These cells were used for FACS analysis to detect early apoptosis using annexin V. After 24 hr culture in the same culture medium and plates, these cells were also collected and analyzed by FACSCalibur Hg flowcytometer. The % of cells showing the fluorescein intensity at M1 region was also calculated and was considered as positive % of apoptotic cells (Fig. 51.2).8

2.6. Caspase-3 Activity

At the indicated time, caspase-3 activity was assayed by caspase-3 activity assay kit (Oncogene (Red-DEVD-FMK) with FACS.

2.7. Extraction of mRNA, cDNA Generation, Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR), Sequencing and Real-Time PCR

Extraction of mRNA and cDNA generatgion was performed. PCR, and Real-time PCR were carried out as reported previously.6 We examined the gene expression of p-53 and b-actin.

2.8. Microscopic and Electron Microscopic Examination

After 24 hour culture, the sample was dissected, postfixed in phosphate-buffered 1% osmium tetroxide (pH 7.4), and dehydrated in an ascending series of ethanol solutions, and observed with a TEM-100CX.

4. DISCUSSION

The signal transduction from extracellular matrix and some soluble factors play an important role for epithelilal cell survival.9 Further the extracellular matrix derived from the RPE is reported to be superior to other material such as tissue culture plastic plate or ligands including gelatin or fibronectin.4 When we collected the RPE from PIPAAm-grafted culture plates, we found more chondroitin sulfate and collagen type IV.

When we examined the RPE as cell sheet from PIPAAm-grafted culture plate, early apoptotic cell death and late cell death were less than those of cells from standard culture plates. Our results may show that rat RPE from PIPAAm-grafted plates as cell sheet may offer better RPE for transplantation than that of standard culture.

5. ACKNOWLEDGMENT

This work was supported in part by grants from Grant-in-Aid for Scientific Research 14370553 (Dr. T. Abe); from the Ministry of Education and Culture of the Japanease Government, Tokyo (Dr. Tamai).

6. REFERENCES

1.LaVail MW, Yasumura D, Matthes MT, et al. Protection of mouse photoreceptors by survival factors in retinal degenerations. Invest Ophthalmol Vis Sci. 1998; 39:592-602.

2.LaVail MM, Unoki K, Yasumura D, et al. Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light. Proc Natl Acad Sci U S A. 1992; 89:11249-11253.

3.Kano T, Abe T, Tomita H, et al. Protective Effect against Ischemia and Light Damage of Iris Pigment Epithelial Cells Transfected with the BDNF Gene. Invest Ophthalmol Vis Sci. 2002; 43:3744-3753.

4.Tezel TH, Del Priore LV. Repopulation of different layers of host human Bruch’s membrane by retinal pigment epithelial cell grafts. Invest Ophthalmol Vis Sci. 1999; 40:767-774.

5.von Recum H, Kikuchi A, Yamato M, et al. Growth factor and matrix molecules preserve cell function on thermally responsive culture surfaces. Tissue Eng. 1999; 5:251-265.

6.Abe T, Sugano E, Saigo Y, Tamai M. Interleukin-1beta and Barrier Function of Retinal Pigment Epithelial Cells (ARPE-19): Aberrant Expression of Junctional Complex Molecules. Invest Ophthalmol Vis Sci. 2003; 44:40974104.

7.Tezel TH, Del Priore LV. Reattachment to a substrate prevents apoptosis of human retinal pigment epithelium.

Graefes Arch Clin Exp Ophthalmol. 1997; 235:41-47.

8.Telford WG, King LE, Fraker PJ. Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell Prolif. 1991; 24:447-59.

9.Prince JM, Klinowska TC, Marshman E, et al. Cell-matrix interactions during development and apoptosis of the mouse mammary gland in vivo. Dev Dyn. 2002; 223:497-516.

Retinal transplantation is based on the hypothesis that the degenerated retina can be repaired by newly introduced normal RPE and photoreceptor cells that may develop appropriate connections with the still functional part of the host retina (Aramant and Seiler, 2002; 2004).

3. RODENT MODELS OF RETINAL DEGENERATION

Retinal degeneration models have been used to test the effects of retinal transplants. The Royal College of Surgeon (RCS) rat expresses a recessive mutation in the receptor tyrosine kinase, Mertk (D’Cruz et al., 2000) which causes RPE dysfunction with subsequent photoreceptor death. In the rd mouse, a mutation of the rod-specific cGMPphosphodiesterase (Bowes et al., 1990; Pittler and Baehr, 1991) results in extensive and rapid loss of incompletely formed rods.

Many transgenic rodent models for various retinal degenerative diseases have been developed. La Vail et al., have created different lines of transgenic rats, carrying either the P23H or the S334ter rhodopsin mutation (Steinberg et al., 1997; Liu et al., 1999; Lee et al., 2003). Many more transgenic mouse models are available for various retinal degenerative diseases (Fauser et al., 2002; Wilson and Wensel, 2003).

Excessive absorption of photons by the visual pigment rhodopsin triggers damage to the photoreceptors (Boulton et al., 2001). Moderate continuous blue light exposure for 2-4 days effectively destroys photoreceptors in albino rats while keeping the RPE intact (Seiler et al., 2000).

4. FUNCTIONAL EVALUATION OF VISUAL RESPONSES

Functional evaluation of visual responses is important for assessment of the efficacy of various treatment strategies in animal models of retinal degeneration.

4.1. Fullfield ERG

One of the first methods used to assess visual function has been the ERG (Jiang and Hamasaki, 1994). However, because the ERG reflects the response of the entire retina, it cannot be recommended for evaluating local retinal responses. As a mass response, the ERG averages the rescued area with much larger areas of nonfunctional retina making it difficult to distinguish the response from the rescued area. For various therapeutic interventions, however, functional recovery may be restricted to specific retinal areas.

4.2. Multifocal ERG

The mfERG has been developed to obtain local retinal responses in patients, and has also been used to detect local functional differences in rats (Ball and Petry, 2000) and mice (Nusinowitz et al., 1999). A more advanced mfERG system is now available powered with a fundus camera that may help to locate responses of specific areas in the retina.

4.3. Recording from Visual Brain Centers

Electrophysiological recording from the visual centers such as superior colliculus (SC) and visual cortex can provide an in-depth analysis of response properties at various levels of the visual centers in the central nervous system (CNS). Recording from the SC may be considered as most effective for the evaluation of the visual responses. The surface of the superior colliculus can be easily exposed and maintained for several hours of recording. Different areas of the retina are represented by corresponding areas on the surface of the SC in a retino-collicular map (Siminoff et al., 1966). In rodents, the SC responses represent mostly direct input from the retina and reflect overall ganglion cell output (Lund et al., 2001). Evaluation of response characteristics such as the response onset latency (Thomas et al., 2004a) may reflect the physiological status of the neural retina. Recording from higher visual areas such as the visual cortex (Girman et al., 1999; Coffey et al., 2002; Girman et al., 2003b) may elucidate the adaptive mechanisms that may occur following visual loss and after therapeutic events aimed at improving visual function.

4.4. Visual Behavioral Tests

Behavioral testing for visual responses which are mediated through the central neural circuitry is an equally important approach for evaluating visual function. Various tests have been employed in rodents to evaluate progression of visual loss following retinal degeneration, and to assess the functional effects of various therapeutic interventions (Lund et al., 2001). These include simple startle reflex (del Cerro et al., 1995) and orientation tests (Hetherington et al., 2000), as well as more complex light discrimination and maze tests (Little et al., 1998; Kwan et al., 1999; Prusky et al., 2000; Coffey et al., 2002). Another particularly effective test of visual performance measures an animal’s ability to track moving stimuli. This head-tracking (HT) test is based on the optokinetic response, a compensatory eye movement in the direction of the movement of a stimulus. By scoring the total time spent tracking the movements, visual acuity can be measured (Coffey et al., 2002). However this technique is not reliable in albino rats due to their abnormal visual sensory apparatus (Precht and Cazin, 1979).

5. RETINAL TRANSPLANTATION

Retinal transplantation, the transplantation of new photoreceptors either as dissociated cells or retinal sheets into the subretinal space, is aimed at replacing the lost photoreceptors (reviewed in Aramant and Seiler, 2002; Lund et al., 2003; Aramant and Seiler, 2004). Intact sheets of fetal retina, with or without RPE, can be transplanted to the subretinal space in various rat models of retinal degeneration and morphologically repair an area of a damaged retina. (Seiler and Aramant, 1998; Aramant et al., 1999) These transplanted photoreceptors can also respond to light (Seiler et al., 1999).

5.1. Restoration of Visual Responses in the SC by Retinal Sheet Transplants in Various Rodent Models of Retinal Degeneration

It was shown in RCS rats that after transplantation of retina together with RPE, visually evoked multi-unit responses could be recorded in the superior colliculus (SC) from 66%

of the transplanted rats, but only from 46% of sham surgery controls (Woch et al., 2001). Transplant responses were only found in a small area of the SC corresponding to the retinal placement of the transplant. Thus if a rescue effect was the mechanism, it appeared to be highly localized. None of the age-matched RCS rats without surgery showed visual responses. It is important to note that a sham surgery effect in RCS rats has been reported previously (Wen et al., 1995; Humphrey et al., 1997). Visual responses of the transplant rats, however, were more robust, and had shorter latencies and higher amplitudes compared with the sham animals. This study indicated that the visual responses recorded from the transplanted rats originated from the transplant area; the possibility of functional connections between the transplant and the host retina was suggested (Woch et al., 2001).

Because the decline in visually driven activity in the RCS rat is slow (Sauve et al., 2001), another degeneration model was selected to better distinguish the transplant-induced recovery/restoration (Sagdullaev et al., 2003). In S334ter-line-3 rats, at the age of transplantation (21-28 days), no visual responses could be recorded within the area of the SC that represents transplant placement. The percentage of transgenic rats with transplants that recovered visual activity (64%) was similar to the percentage reported for RCS rats (66%). Again, visual responses were recorded only from a small area of the SC corresponding to the retinal placement of the graft. None of the age matched control rats including the sham surgery rats showed visual responses in the SC. Qualitative anatomical comparisons suggested that one important predictor of functional outcome is the morphological integrity of the transplant. The lack of a sham surgery effect in S334ter-line 3 rats may be attributed to the difference in the mechanism underlying retinal degeneration in different degeneration models (RCS and line-3 transgenic rats).

To study the long term effect of retinal transplantation, visual responses were recorded from S334ter-line-5 rats, a model for slow retinal degeneration (Thomas et al., 2004a).Visual responses can persist for a substantial period of time after neural retinal transplantation because these responses are preserved in the transplanted area of line-5 rats up to 254 days of age. However, none of the “poor” or disorganized transplants had any effect. Further, all the rats with “good” transplants had relatively “short” latency responses suggesting better inner retinal function.

Retinal transplantation studies conducted in rd mice (Arai et al., 2004) yielded a 43% success rate (positive responses were recorded from three out of seven eyes studied). Interestingly, the organization of the graft did not appear to correlate as expected with the electrophysiology results in this model. Eyes with well-organized, laminated grafts showed no response whereas the three light-responsive eyes had rosetted or disorganized grafts. All three light-responsive eyes demonstrated much higher levels of recoverin immunoreactivity in the host retina overlying the graft compared with untreated age-matched rd/rd mice. Thus, the mechanism of visual restoration after retinal sheet transplantation in this experiment appeared to derive at least in part from a rescue effect on host cones. The lack of an apparent functional rescue effect in the animals with laminated grafts may have been due to a glial barrier between transplant and host, and the loss of cones in the host retina.

5.2. Better Head Tracking Response in Transplanted S334ter-Line-3 Rats

The functional capability of retinal transplants in S334ter-line-3 rats was further evaluated by means of optokinetic behavioral tests, a modified optokinetic head tracking apparatus (Thomas et al., 2004b). This apparatus consists of a striped rotating drum, specifically

Figure 52.1. Optokinetic Testing (modified method to test each eye separately) of S334-ter-line 3 rats, 166236 days old. The head tracking score is compared between left and right eye among transplanted S334ter-3 rats. Significant preservation of visual responses was seen in the transplanted eyes at later stages of retinal degeneration. Among non-transplanted rats the progression of visual loss is more symmetrical in both eyes. Taken from Figure 5A of Thomas et al., 2004b. Optokinetic test to evaluate visual acuity of each eye separately. (Reprinted from Journal of Neuroscience Methods, Vol 138, Thomas et al., Optokinetic test to evaluate visual activity acuity of each eye separately, 7-13; 2004, with permission from Elsevier).

modified to measure vision in each eye separately for evaluation of monocular treatments. In line-3 rats with retinal transplants, visual responses are significantly preserved in transplanted eyes at late stages of retinal degeneration (Fig. 52.1). The result of the behavioral evaluation corroborates our electrophysiological findings and demonstrates that the visual sensitivity of transplant is more than just light perception.

5.3. Transplanted S334ter-Line-3 Rats Show Improved Visual Sensitivity to Low Light

The light stimulus for the above SC recordings in S334ter-line-3 rats (Sagdullaev et al., 2003) consisted of a full-field stimulus with bright light of 1300 cd/m2 (3.11 log cd/m2). In the following experiments, the recording stimulus was modified to evaluate the sensitivity of the visual responses (visual threshold). Light stimuli of 100 ms to 1 second duration and intensity -8 to 1 log cd/m2 were presented on a full field white screen. -6 log cd/m2 corresponds to the rod threshold (overcast night sky), -1 log cd/m2 corresponds to the cone threshold (dawn). Visual responses from the SC were recorded from pigmented normal as well as retinal degenerate rats (with or without transplants). In normal pigmented rats, the dark adapted visual threshold was -5.52 log cd/m2 which is almost similar to previously reported observations in pigmented Long Evans rats (Herreros de Tejada et al., 1992). In transgenic retinal degenerate rats, at 45 days of age, the threshold was elevated to -0.09 log cd/m2. On the other hand, the visual threshold in the SC was considerably improved in transplanted line-3 rats (Fig. 52.2) in the area corresponding to the placement of the graft in the retina.

Girman et al. (2003a) demonstrated that the mesopic range of normal pigmented rats is at around -4 log cd/m2. Once it is possible to determine the scotopic and photopic range of dark adapted rats, it is also possible to assess whether the visual responses originate from

Latency (ms)

Figure 52.2. Visual responses recorded from the superior colliculus of transplanted rats after low light stimulation. Transplanted rats show similar latency responses as normals down to a stimulus intensity of -3 log cd/m2 in the SC area corresponding to the graft placement. No surgery S334ter-3 rats, which contain no rods, show only long latency responses to bright light, and no responses to low light (below 0 log cd/m2).

the host photoreceptors because the rods are only found in the transplants and not in the host retina. Preliminary data suggest that visual threshold recorded in the transplanted line- 3 rats is above the level of a clear rod response. However, the possibility of rod contribution cannot be ruled out for several reasons. The physiological capability of the transplanted photoreceptors may not be expected to be same as normal rods. Also, the neural circuitry formed between the transplant and host retina may influence the light threshold level of the transplant. Also, it should be noted that in a non-transplant rat, the visual threshold at a very early age (45 days) is much higher (-0.09 log cd/m2) than the threshold recorded from a transplanted rat. This suggests that restored visual response is not due to photoreceptor rescue but a more direct contribution of the transplanted photoreceptors.

6. ADVANTAGES OF FETAL RETINAL SHEET TRANSPLANTS

Although there are other important treatment strategies aimed at improving visual function, retinal transplantation remains a major hope for those who have lost the photoreceptors. Fetal retinal sheet transplants with or without RPE, can morphologically integrate into a degenerated retina. This has been demonstrated in various retinal degeneration rat models (Seiler and Aramant, 1998; Aramant et al., 1999; Woch et al., 2001; Sagdullaev et al., 2003; Thomas et al., 2004a). Significant improvement in visual sensitivity has been reported following transplantation of a fetal retinal sheet together with its RPE in a human patient (Radtke et al., 2004). This patient improved from hand motion vision to being able to read enlarged letters from a computer screen.

The various advantages of fetal retinal sheet transplantation technique are briefly described here.

6.1. Transplants Reconstruct the Damaged Retina

Using a special implantation instrument and procedure developed by Aramant and Seiler (Aramant and Seiler, 2002), it is possible to transplant fresh intact sheets consisting of 2 tissues: RPE together with the neuroblastic retina. The photoreceptors of intact-sheet transplants develop both inner and outer segments morphologically resembling a normal retina and remain healthy for many months (Seiler and Aramant, 1998; Aramant et al., 1999; Seiler et al., 1999). These transplanted sheets of retinal neuroblastic progenitor cells already have a primordial circuitry established.

6.2. Transplants are Physiologically Active

It has been shown that normal phototransduction processes takes place in the transplanted photoreceptors (Seiler et al., 1999) which are hence capable of transforming light into electrical signals. There are also indications to prove establishment of neural connections between the transplant and the host retina (Seiler et al., in press).

6.3. Trophic Effects of Fetal Retinal Sheets

It has been well established that trophic factors can be used as a treatment strategy to protect photoreceptors from degeneration (Delyfer et al., 2004). Most of the trophic factors are naturally present in a normal healthy retinal environment. Following cell loss, the natural resources for these trophic factors will be depleted and subsequently a degenerating retina is subjected to large-scale secondary changes. Supplementation with one single factor may not be of sufficient help to reverse these changes. The fetal retinal sheets, especially when transplanted together with its RPE can be a good source of many different trophic factors. Hence, fetal retinal sheet transplantation may help to regain the normal retinal homeostasis by protecting the inner retina from further damage.

6.4. Permanence of the Transplants

For any therapeutic strategy, it is very important that the functional recovery is longlasting. A significant advantage of the fetal retinal transplants is that they can remain safely in the host environment for an extended period of time. This may be due to the relative immunological privilege of the subretinal space. The safety of the transplants in the subretinal space has been shown in animal models (Thomas et al., 2004a) as well as in human patients (Radtke et al., 2002; Radtke et al., 2004). The functional effects of transplants can be maintained through a large part of the life span of the experimental animals (Woch et al., 2001; Sagdullaev et al., 2003; Thomas et al., 2004a). A report from a clinical trial showed improvement in visual sensation in some patients even years after surgery (Radtke et al., 2004).

6.5. Transplants Provide Mechanical Support to the Degenerating Retina

There are recent reports on elaborate changes taking place in the retina following degeneration (Jones et al., 2003; Marc and Jones, 2003; Marc et al., 2003). The loss of photoreceptors and the subsequent change in the retinal thickness can be one of the factors that