0042-6989/92 $5.00 + 0.00 Copyright 0 1992 Pergamon Press plc

Vision Res. Vol. 32, No. I, pp. 37-40, 1992 Printed in Great Britain. All rights reserved

Magnetic Resonance Microscopy of Rabbit Eyes AKITOSHI YOSHIDA,*.t KENNETH K. KWONG,**$ CHEN CHANG,$ KOUSEE KARINO,* TAKUYA IWASAKI,? SHELDON M. BUZNEY,? J. WALLACE McMEEL,t HONG-MING CHENG*§ Received 25 February 1991

Magnetic resonance (MR) micro-imaging was performed on enucleated eyes from rabbits previously injected with perfluoropropane gas (CT8), with or without the surgical creation of retinal detachment. Condensed vitreous, which exhibited shortened longitudinal relaxation time (Tl), could be d@erentiated with proton-density and Tl-weighted imaging. Gradient-echo imaging could in addition detect vitreo-retinal tractions. The detached retina itself was also seen. Further, proton-density but not TI -weighted imaging showed lens opacities appearing as high-intensity regions. MR microscopy is a convenient method for gross morphological examination of intact eyes.

MR microscopy Rabbit eye CJF, injection Condensed vitreous Vitreous traction ment Lens opacity

INTRODUCTION

the magnetic field strength. For example, imaging at 8.69.4 T with an in-plane resolution of 10 pm has been attained (Aguayo et al., 1986, 1987; Aguayo & Cheng, 1987). To exploit this method and to visualize several common ocular disorders, we examined rabbit eyes previously injected with perfluoropropane (C,F,) gas and eyes with surgically induced retinal detachment, and the results are reported here.

Proton magnetic resonance (MR) imaging has become an important tool in ophthalmic research. Both anatomical and biophysical information of ocular tissues can be obtained from intact eyes. Several studies have investigated vitreous changes following enzyme digestion and pathologies in enucleated eyes (Gonzalez, Cheng, Barnett, Aguayo, Glaser, Rosen, Burt & Brady, 1984; Aguayo, Glaser, Mildvan, Cheng, Gonzalez & Brady, 1985; Miglior, Kain, Libondi, Gonzalez, Barnett, Krauss & Cheng, 1986; TerPenning, Cheng, Barnett, Seddon, Sang, Latina, Aguayo, Gonzalez & Brady, 1986). These studies were conducted at 1.4 T; image resolution at this and similar magnetic field strengths (usually >0.4 mm in-plane resolution) is inadequate for the analysis of morphological details. A powerful extension of MR imaging, MR microscopy, recently has been developed and used to examine implanted tumors in mice, enucleated rodent eyes (Aguayo, Blackband, Schoeniger, Mattingly & Hintermann, 1986; Aguayo, Blackband, Wehrle & Glickson, 1987; Aguayo & Cheng, 1987) and galactose cataract in rabbits (Ahn, Anderson, Juh, Kim, Garner & Cho, 1989). MR microscopy can be achieved by increasing the strength of the field gradients and/or

MATERIALS AND METHODS New Zealand white and pigmented rabbits of both sexes weighing 2.5-3.0 kg were used in this study. Anesthesia consisted of an intramuscular injection of ketamine (200 mg/kg) and chlorpromazine (25 mg/kg) together with 0.5% proparacaine eye drops. Both pupils were dilated with 10% phenylephrine and 1% tropicamide eye drops, the eyes were examined by biomicroscopy and indirect ophthalmoscopy to ensure the absence of abnormalities. The rabbits (N = 5) were restrained in a dorsal position. 0.4 ml of 100% C,F, (PCR Research Chemicals, Gainesville, Fla) was injected in the right eye through a sterile 0.45 micron filter (Millipore, Bedford, Mass.) attached to a 30-gauge needle into the center of the vitreous cavity, 1 mm from the cornea1 limbus as described by Iwasaki, Buzney & Hirose (1988) and Iwasaki, Buzney, Seery, Ueno & Davison (1989). The purpose of gas injection was to compress the primary vitreous thus causing vitreal condensation. In another group of rabbits (N = lo), a retinal hole was created in the right eye four days after gas injection. The purpose was to induce retinal detachment. Briefly, the rabbits were anesthetized in the same way described

*Howe Laboratory of Ophthalmology, Harvard Medical School, and the Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, U.S.A. tThe Eye Research Institute of Retina Foundation. SDepartment of Radiology, Harvard Medical School, Boston, MA 02114, U.S.A. §To whom reprint requests should be addressed. Dr Yoshida is on leave from the Department of Ophthalmology, Asahikawa Medical College, Asahikawa 078, Japan. YR32,I-c

Retinal detach-

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above and were secured on the operating table in a lateral position. A peritomy was created at the l&l 1 o’clock meridian and a sclerotomy site was prepared 1 mm from the corneoscleral limbus. Using a surgical contact lens (Mira, Waltham, Mass.) and an operating microscope (Zeiss) to guide the needle, the previously injected C,F, bubble was aspirated from the vitreous through this sclerotomy site via a 25-gauge needle and the gas was replaced with balanced salt solution (BSS, Akron, Abita Springs, La). A blunt 26-gauge cannula (Storz) was inserted through the sclerotomy and used to gently aspirate a 3-5 disc dia area of the retina immediately beneath the optic disc, thus creating a retinal hole. The sites of the sclerotomy and peritomy were closed with separate 8&O nylon sutures. The rabbits were examined periodically with biomicroscopy and indirect ophthalmoscopy to record the occurrence and progression of ocular changes. Rabbits with evidence of extensive ocular changes were selected for the MR study. They were sacrificed with an overdose of i.v. pentobarbital and the eyes enucleated. MR microscopy was then performed on the eyes using the method of Aguayo and Cheng (1987) with a Bruker MSL400 (9.4 T; 89 mm bore size) NMR spectrometer. The eyes were placed in a 20 mm NMR tube and lowered into the magnet previously fitted with a 20 mm slotted tube resonator. The pulse sequences and MR parameters are described in the figure legends. Briefly, MR images were acquired according to three main NMR parameters: longitudinal relaxation time (TI), transverse relaxation time (TZ), and proton-density. We used both spin-echo and gradientxcho pulse sequences. In our experience, 7’2-weighted spin-echo ocular images do not show adequate anatomical details as well as those of Tl -weighted and proton-density images, T2-weighted images therefore were not presented. In gradient-echo imaging, the ethos were generated by reversing the field gradient thus allowing faster image acquisition than spinecho imaging. Selection of MR parameters was achieved by using different TRs (repetition times) TEs (echo times), and in gradient-echo imaging, the tip angle in addition. To avoid image fold-over, the readout gradient was adjusted so that the in-plane resolution was fixed at 103 x 206 pm (i.e. with a matrix of 256 x 256 but only 128 phase-encoding steps) and the slice thickness was 1 mm. Further improvement of resolution was possible but only partial images were obtainable. For this study, only full images of the eyes were acquired. Following MR studies, the eyes were placed in Karnovsky’s fixative (2% paraformaldehyde + 2.5% glutaraldehyde) overnight and bisected in a plane that matched the MR imaging plane, and examined under a dissecting microscope (Zeiss). RESULTS

The injected C,F, rapidly expanded and filled most of the vitreal cavity within three days. This immediately caused physical compression of the vitreous gel and in

c( ui

some cases, formation

of lens opacities. The gas bubble then gradually diminished until complete absorption four weeks after injection. During this time, the vitreous re-filled: most eyes demonstrated antero-posterior strands in the vitreous cavity (i.e. condensed vitreous gel) extending from the optic disc and the medullary rays towards the ciliary body (cf. Miller. Lean. Miller Rr Ryan, 1984), and the more liquefied vitreous was ohserved around the condensed vitreous with slip lamp examination. Five weeks following CIF, injection the eyes were enucleated and subjected to MR microscopy. Figure 1 represents a proton-density map of the control eye. The cornea/sclera, anterior chamber, irisjciliary body, lens, and vitreous cavity are clearly seen which are free of‘ any pathology. Figure 2 shows a proton-density image of an eye with previous C,F, injection. The gas had already been totally resorbed (its presence was indicated by a signal-void region, results not shown). The lens demonstrated a high-intensity area deep in the posterior cortex corresponding to the supranuclear region. Three other high-intensity vitreous regions immediately posterior to the lens are also seen. These regions correspond to the condensed vitreous gel later confirmed with optical microscopy. Tl-weighted images also showed the three vitreal regions but not lens opacities (Fig. 3). T_?weighted images, on the other hand, showed no significant difference between the gas injected and the control eyes. Two days after creation of the retinal hole, biomicroscopy and indirect ophthalmoscopy revealed that the edge of the retinal hole was rolled and averted. Subsequently, all eyes with total or partial retinal detachment developed some degree of proliferative vitreoretinopathy (PVR)-like changes as described by Iwasaki et al. (1988, 1989). These included vitreous strands or membranes, retinal vascular changes, puckers of the medullary wings and fixed retinal folds. Figure 4 shows the proton-density image of an eye with total retinal detachment, four months after surgical creation of the retinal hole. V-shaped retinal detachment was observed in the vitreous cavity. There was also a high-intensity region at the posterior pole of the lens. Figure 5 is a TI -weighted gradient-echo image demonstrating fine fibrillar-like structures in the vitreous cavity between the detached most likely represent be observed optically eyes.

retina and the lens. These “striae” vitreoretinal tractions that cannot and are not detected in the control

DISCUSSION The eyeglobes used in this study were imaged immediately after enucleation and delayed postmortem changes were not investigated. Further studies are needed to establish the validity of MR microscopy particularly its correlation with histological analysis. Morphologically, the lens can be separated into highand low-proton-density regions (Fig. 1) that correspond to the anatomical cortex and nucleus, respectively. The

MR

FIGURE FIGURE

1. A proton-density

OF

FIGURE

RABBIT

EYES

image of the normal control eye. The repetition time (TR) was 4 set and the echo time (TE) 10 msec. The imaging time was 17 min with two averages.

2. Proton-density image of an eye previously injected cortical (supranuclear) cataract and condensed vitreous.

FIGURE condensed

FIGURE

MICROSCOPY

with C,F, (TR = 3 set; TE = 10 msec) showing posterior C,F, has been totally resorbed prior to imaging.

3. A TI-weighted image of the same eye as in Fig. 2 (TR = 300 msec; TE = 10 msec). Notice appearance of the vitreous but not the lens opacities. The imaging time was 11 min with 16 averages. The image quality appears poor because the short TR limited signal recovery. 4. A proton-density

image

of a rabbit

eye (TR = 4 set; TE = 10 msec) showing

the detached

retina

(arrows).

5. A gradientecho image (TR = 300 msec; TE = 4 msec; tip angle = 30”) showing striae or vitreous traction the lens and the detached retina next to the posterior pole. The scan time was 1.5 min with 2 averages.

between

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YOSHIDA et nl

lens nucleus contains not only less water but also has much shorter TZs than the cortex, it therefore appears dark (Cheng, Yeh, Barnett, Miglior, Eagon, GonzSez & Brady, 1987; Aguayo & Cheng, 1987). Both the supranuclear cataract (Fig. 2) and the posterior cortical cataract (Fig. 4) contain the highest water content in the lens which may be an indication of cellular disintegration. It is interesting to note that the cataracts remained discrete suggesting an absence of bulk water movement. Condensed vitreous gel can be clearly seen with MR microscopy: proton-density images showed enhancement in the anterior vitreous which matched the same area of high condensation later revealed by gross optical examination. Unlike lens opacities (Fig. 2), this result indicates that the condensed vitreous gel has shortened Tl s. This is in agreement with TerPenning et al. (1986) who reported shorter TI values in vitreous condensation (asteroid hyalosis) in post-mortem diabetic eyes. Injection of the C3F, may cause molecular changes in the vitreous gel (Thresher, Ehrenberg & Machemer, 1984). However, the gas expansion process is often accompanied by secondary inflammations. These inflammation processes manifested as “vitreous haze” and “fibrillar debris” in the vitreous gel. The condensed vitreous gel observed in the gas injected eye may result from both the molecular change and the mechanical force. In any case, the gel has apparently become less water-soluble causing a shortening of TI s. The proton-density image clearly demonstrated the detached retina (Fig. 4) which has not been previously detected with low-field clinical imaging. The enhancement in contrast observed with gradientecho imaging is due to the magnetic susceptibility or boundary effects. This method can detect fine vitreous changes such as vitreoretinal tractions as seen in Fig. 5. We know of no other methods capable of detecting these changes. Our study demonstrates that common lesions in the eye such as retinal detachment, cataracts, and vitreous condensation can be effectively examined with MR microscopy. Future development will include in vivo MR microscopy which is possible with increasing gradients (e.g. by using surface gradient coils) to improve resolution and surface radio-frequency coils to improve signal-to-noise. However, the eye motion may remain a source of interference on image quality. Gated imaging or image editing will be required. With the wide variety of pulse sequences that can be performed in the threedimensional mode, this method can become a valuable

diagnostic tool especially in cases where optical observation is impeded by opaque media or when uttrasonographic data appear nebulous.

REFERENCES Aguayo, J. B. & Cheng, H. M. (1987). Magnetic resonance microscopy of ocular tissues. Medical Science Research, 15, 10.59-1060. Aguayo, J. B., Blackband, S. J., Wehrle, J. P. & Ghckson, 3. D. (1987). NMR microscopic studies of eyes and tumors with histological correlation. Annals of the New York Acaa’emy yf Sciences, 508. 399413. Aguayo, J. B., Blackband, S. T., Schoeniger, J., Mattingly, M. A. & Hintermann, M. (1986). NucIear magnetic resonance ima&g of a single cell. Nature, 32.2, 190-191. Aguayo, J., Glaser, B., Mildvan, A., Cheng, H. M., Gonz&lez, R. G. & Brady, T. (1985). Study of vitreous liquefaction by NMR spectroscopy and imaging. Investigative Ophthalmology and Visual Science, 26, 692691. Ahn, C. B., Anderson, J. A., Juh, S. C., Kim, I., Garner, W. H. L Cho, Z.-H. (1989). Nuclear magnetic resonance microscopic ocular imaging for the detection of early-stage cataract. ~nvestjgatil,~ OFhthaImalagy

and Visual Science, 30, f612-1617.

Cheng, H. M., Yeh, L. I., Batnett, P., Miglior, S., Eagon, J. C., Gonzalez, R. G. & Brady, T. J. (1987). Proton magnetic resonance imaging of the ocular lens. Experimental Eye Research, 45,875.882. Gonzalez, R. G., Cheng, H. M., Barnett, P., Aguayo, J. B., Glaser, B., Rosen, B., Burt, C. T. & Brady, T. (1984). Nuclear magnetic resonance imaging of the vitreous body. Se&tee, 223, 399-400. Iwasaki, T., Buzney, S. M. & Hirose, T. (1988). PVR following experimental rhegmatogenous retinal detachment in rabbits, Inve..rtigative Ophthalmology

and Visual Science, 29 (SuppI.), 305.

Iwasaki, T., Buzney, S. M., Seery, C., Ueno, N. L Davison, P. (1989). Altered matrix in experimental PVR. Investigative Ophthalmology and Visual Science, 30 @up&.),

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Miglior, S., Kain, H. L., Libondi, T., Gontilez, R. G., Barnett, P. A., Krauss, J. M. & Cheng, H. M. (1986). Early vitreous changes in experimental proliferative vitreotinopathy. Ar&

Magnetic resonance microscopy of rabbit eyes.

Magnetic resonance (MR) micro-imaging was performed on enucleated eyes from rabbits previously injected with perfluoropropane gas (C3F8), with or with...
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