(Accepted 27 August 1986)

Neuroanatomy of the human visual system: Part I Retinal projections to the LGN and pretectum as demonstrated with a

new method

ALFRED0 A. SADUN*

Departments of Ophthalmology and Neurosurgery, USC School of Medicine, Los Angeles, CA, USA

ABSTRACT. The neuroanatomy of experimental animals has been extensively investigated with studies which have employed sophisticated histological and physiological techniques. However, there has been very little information on human neuroanatorny since these techniques have been largely inapplicable to human brains. In particular, silver staining techniques have not been as successfully applied in human brains since they require precise, short survival times.

A staining method (PPD) has been developed which permits the tracing of the degenerated fibers in the human brain even after very long survival periods. Applying this method to postrnor- tem brains from patients with old ocular pathology, the author was able to demonstrate projec- tions from the retina to the lateral geniculate nucleus and the pretectum in man. Transsynaptic changes seen in the lateral geniculate following long-term deafferentation are best described as atrophy, not degeneration. The PPD method is a simple and reliable technique that can be used in conjunction with electron microscopy in delineating the neuroanatomy of the human visual system.

Key words: paraphenylene-diamine ; PPD method ; human neuroanatomy ; visual system ; lateral geniculate nucleus ; pretectum; axonal degeneration

INTRODUCTION

The neuroanatomy of the vertebrate visual sys- tem has been extensively described in experimental animals through investigations that have employed either histological or physio- logical techniques (Polyak, 1957). Silver impreg- nation staining has proved to be a particularly fruitful method, permitting the tracing of

* Reprint requests to: Alfred0 A. Sadun, M.D., Ph.D., Department of Ophthalmology, USC School of Medicine, 1355 San Pablo St., Los Angeles, CA 90033, USA

degenerating fibers produced by experimental lesions. There have been many refinements in the silver impregnation of degenerating axons since 1850, when Augustus Waller showed that nerve transsection resulted in degeneration of the distal segment of that nerve. Improvements made by Nauta & Gygax (1951), Nauta & Ryan (1952), and Fink & Heimer (1967) have permit- ted extensive documentation of degenerating fibers in animal brains. More recently, tech- niques such as retrograde tracing by horseradish peroxidase, have been developed that utilize the axonal transport system ; other immunochemi-

Neuro-ophthalmology - 1986, Vol. 6, No. 6, pp. 353-361 0 Aeolus Press Amsterdam 1986

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A . A . Sadun

cal and electrophysiological techniques comple- ment such methods for investigations in animal brains.

Despite these experimental advances, neu- ropathology has provided surprisingly little information about human neuroanatomy, as the techniques described above have been more or less infeasible for application to human brains. Recently, a few investigators (Mesulam, 1979; Grafe & Leonard, 1980) have, by applying very careful controls in the staining parameters of their own modifications of silver stains, estab- lished some results in finding degeneration of axons in the human brain even after very long- term survivals. These studies, however, were not on retinofugal projections and relied on compli- cated and capricious methods that did not pro- vide high resolution for microscopy. Herein we report a recently developed modification of a staining method that can be used to great advan- tage in studying the human visual pathways (Sadun et al., 1983 ; Sadun & Schaechter, 1985).

Paraphenylene-diamine was utilized by Schultze in 1917 to intensify the contrast in fro- zen sections of animal neural tissue that had been previously osmicated. Estable-Puig el al. (1965) and Hollander & Vaaland (1968) modi- fied the technique for use on tissue embedded in plastic. Hollander & Vaaland (1968) and Sadun (1975) demonstrated in experimental animals that this technique could be used to identify degenerating axons and axon terminals, and that the degeneration could then be confirmed by ultrastructural examination of the same section of tissue (Sadun, 1975). We have further modi- fied this method (PPD) to stain degenerated fibers in the human brain (Sadun er al., 1983).

Wallace (1836) described the course of gan- glion cell axons in the human retina. These axons merge in the anterior nerve head and then course posteriorly to the optic chiasm. The optic

tract is formed by the ipsilateral temporal fibers and contralateral nasal fibers, and appears to terminate predominantly in the lateral genicu- late nucleus (Duke-Elder & Wybar, 1961 ; Kup- fer, 1962; Walsh & Hoyt, 1969). Polyak (1957) and others have proposed that some retinal fibers may terminate in the pretectal region. The PPD method provides the tool to confirm the existence and define the nature of these two retinofugal fiber projections in man.

MATERIALS AND METHODS

Postmortem brain specimens were obtained from monkeys and humans. Three Cynomolo- gus monkeys underwent uniocular enucleation and were sacrificed nine months later under anesthesia (sodium pentobarbital) by perfusion with the following fixative : 2% formaldehyde (from paraformaldehyde), 2% glutaraldehyde, and 2.5% dimethylsulfoxide in 0.1 M sodium cacodylate buffer (pH 7.4). Fifteen human autopsy brains, 12 of which had clinical documentation of ocular or optic nerve damage prior to death, were also used for this study. The etiology of the ocular or optic nerve damage in these patients included retinitis pigmentosa, sur- gical or post-traumatic enucleation, optic nerve glioma, optic nerve sheath meningioma, aneu- rysm of the ophthalmic artery, nasopharyn- gioma, and pituitary adenoma. Most of the lesions resulted in complete atrophy of one or both optic nerves. The survival periods for these cases ranged from six months to 40 years, and the patients ranged in age from 18 to 82 years. Three of the 15 human cases were without clini- cal or histological evidence of damage to the vis- ual system; these brains served as controls.

The postmortem human brain specimens were obtained between four and 24 hours after death; in most instances the brains were then immersed

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Retina/ projecrions to the LGN and pretectum

in 10% formalin solution for several weeks. 1 Specimens were obtained from the optic nerves, 1 optic chiasm, optic tracts, lateral geniculate nucleus (LGN), superior colliculus (SC), and , pretectum (PT). Specimens were cut into blocks '

of approximately 1 cm3; they were then trans- ferred into the buffered fixative described above, and stored at 4°C.

were cut for processing, rinsed in 0.1 M sodium cacodylate buffer (pH 7.4), and incubated over- night in cold 0.5% osmium tetroxide in buffer. The tissue was then rinsed several times in buffer and distilled water, dehydrated through a graded series of alcohols and propylene oxide, and then infiltrated with EMbed 812 (an epoxy resin), The tissue was polymerized in cylindrical capsules at 60°C for about 60 hours. The blocks were then trimmed and semithin (1-2 pm) sections cut on a glass knife. The sections were placed on dis- tilled water drops on glass slides and dried at approximately 90 "C and then allowed to cool to room temperature. The sections were stained by immersing the slides in a solution of 1% paraphenylene-diamine in methanol for five to ten minutes at room temperature. The slides were rinsed in absolute ethanol for five minutes, rinsed again with 95% ethanol, air dried, and cover-slipped. The paraphenylene-diamine solu- tion had been previously oxidized by allowing it to stand at room temperature for three to six weeks, until it became dark brown in color. The staining intensity of the sections did not vary with the duration of staining or rinsing; the darkness of the stained tissue could be con- trolled only by varying the section thickness (Sadun et af., 1983). All sections were stained with only PPD unless otherwise noted.

Ultrastructural verification of degeneration was made on thin sections cut from the same plastic blocks. This was done either by cutting

Slices of tissue approximately 0.5 x 4 x 6 mm I

serial sections, or by removing a 2 to 3 pm thick PPD stained section that had been mounted previously on a glass slide and photographed (by the method of Sadun & Schaechter, 1985). This section was affixed to the end of a blank plastic block prior to further cutting. Thin sections were placed on copper grids and stained with uranyl acetate and lead citrate according to the Reynold's method (1963). The grids were then examined with a Philips 300 or Zeiss EM-10 elec- tron microscope.

RESULTS

Sections from the optic nerves, optic tracts, LGN, and PT were examined with the PPD met hod.

Monkey

The optic nerve contralateral to the lesion was seen in cross section to be composed of tightly packed dark annuli. The myelin ring of each normal axon appeared as a darkly stained annu- lus ; the cytoplasm remained unstained. Exami- nation of the optic nerve ipsilateral to enuclea- tion showed circular profiles with dark contents, indicative of axonal degeneration ; normal axons with typical myelin ring ensheathment were not seen. Axonal degeneration was also noted bilaterally in sections of the optic tracts (Fig. la), LGN (Fig. lb), and PT (Fig. lc). Ultrastructural examination from the same blocks confirmed the presence of degenerated axons and axon preterminals (Fig. 2).

Man

Cross sections through undamaged human optic nerves revealed clusters of dark rings indicative of normal myelinated axons (Fig. 3a). Degener-

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A. A. Sndun

Fig. I . a. Cross section through monkey optic tract from an animal with optic nervedamage; degenerated axons (arrows) are present ( X 850, PPD). b. Section of monkey lateral geniculate nucleus containing degenerated axons (arrows) ( X 850, PPD). c. Section of monkey pretectum containing degenerated axons (arrows) (x 850, PPD).

ation consisting of darkly stained circular pro- files (Fig. 3b) was noted in the optic nerves that had suffered a lesion or that were ipsilateral to ocular lesions. Small clusters of degenerated fibers were also seen interspersed with normal axons in the optic chiasm and in the optic tract. Examination of the optic nerves, chiasms, and tracts from the normal (control) human brains showed clusters of tightly packed, darkly stained myelin rings. Few, if any, degenerated fibers were seen.

Sections of the LGN from cases in which there had been prior damage to the optic nerves were examined, revealing degenerated axons and degenerated axon preterminals (Fig. 4a). The

layers of the LGN that received fibers from the intact eye (layers 2, 3, and 5 in the LGN ipsilateral to the normal optic nerve) had fewer degenerated axons than did those layers (1, 4, and 6) that received fibers from the damaged eye. Transsynaptic atrophy of the large neurons was also noted in the cases with survival periods exceeding two years (Fig. 4b). Neither trans- synaptic changes nor degeneration were noted in the normal (control) human brains.

Sections were also examined from an area dorsal to the LGN, which contains the optic radiations emanating from the LGN. Degenera- tion was not seen in any of the cases with optic nerve damage even in cases that showed con-

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Retinal projections to the LGN and pretectum

Fig. 2. Electron micrograph of monkey lateral geniculate nucleus, layer 1, following contralateral optic nerve damage. Normal axons (N) are present, as are degenerated axons (D: ( x 23,500).

siderable transsynaptic cellular atrophy in the LGN .

A number of degenerated fibers were also noted immediately medial to the LGN. These fibers coursed past the medial geniculate nucleus and then appeared to enter the brachium of the SC. Degeneration was also noted profusely throughout the pretectal area. Degenerated axons and axon preterminals were seen bilaterally in the F'T in human cases with prior unilateral optic nerve damage (Fig. 5) .

DISCUSSION

The recently developed PPD method permitted

Fig. 3. a. Cross-section of normal human optic nerve shows tightly packed myelin rings of retinal ganglion cell axons ( x 1000, PPD). b. Cross-section of damaged human optic nerve (survival rime: one year) ; degenerated axons (arrows) are present ( x 1000, PPD).

us to identify and follow degenerated axons in the human brain. Degeneration was traced from the eye through the ipsilateral optic nerve, and through both optic tracts. Occasionally, degenerated axons were seen in tissue collected from patients with normal clinical histories. We examined this phenomenon in tissue from very young to very old patients, and attribute it to background cell death. Background degenera- tion was seen to increase with increasing patient age. Retinofugal projections were found in the LGN and F'T. Ultrastructural examination con- firmed the presence of long-standing products of degeneration. The PPD staining method thus

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Fig. 4. a. Section of human lateral geniculate nucleus (survival time: one year), layer 2, containing degenerated axons (arrows) (x 400, PPD). b. Human lateral geniculate nucleus. Cell bodies were innervated by ipsilateral eye; laminae 1, 4, and 6 contain atrophic cell bodies (a) (H&E, x 20).

proved to be a reliable light microscopic method to identify degenerated axons and even axon preterminals in human tissue.

Kupfer (1965) and others have described changes in cell size and number in those lamina of the LGN that received projections from an injured eye. Polyak (1957) concluded from clini- cal pathological correlations that there must be a projection from the retina to the pretectal region of the human brain. The present study histologically confirms these two pathways.

Like Kupfer (1965), we noted transsynaptic atrophy among LGN neurons in cases in which the optic nerve damage had existed for more than two years. The term atrophy, and not degeneration, must be emphasized for two rea-

sons: first of all, most of the neurons in the atrophied layer of the LGN were still present. While cell counts are often inaccurate due to tis- sue shrinkage, variability in the plane of section and other reasons, we did not note obvious decreases in cell numbers in any of the layers of the LGN. Additionally, the term degeneration should be avoided since no degeneration was noted in the optic radiations emanating from an LGN densely populated with degenerated axon terminals and with marked transneuronal atro- phy. In cases of long term optic atrophy, degenerated axon terminals have been observed in layers 4b and 4c of the visual cortex; yet axons in the optic radiations are not degenerated more proximally (Smith et af., 1982). The limited

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effect of retinal deafferentation was shrinkage of the postsynaptic element (atrophy) but not cellular death (degeneration). Degeneration found in the brain following an ocular lesion is thus directly attributable to that lesion, and is not a consequence of a series of transsynaptic degenerations.

Several obstacles have prevented effective histological study of autopsy brains with long- standing lesions as a method of directly study- ing human visual neuroanatomy. Lesions produced by injury or disease are not always well delineated and survival periods are variable and almost always prolonged, usually exceeding the very transient period of active fibrillar degeneration that can be stained by silver impregnation methods. Fixed human material is almost always of poor quality since perfusion is precluded and there is usually a long period between death and autopsy/tissue fixation. Con- trols are difficult to establish whenever dealing with human cases; the lesions of nature are often irregular and not reproducible, therapeutic interventions may obscure the extent of damage, and selection biases must be addressed. The additional problem of an intrinsic difference in the staining quality of human brain tissue has further handicapped attempts at direct histolog- ical delineations of human visual anatomy. Polyak’s review of the vertebrate visual system relies in part on Gratiolet for description of the human visual pathways (1957). Gratiolet’s (1854) methodology was to use orangewood sticks to dissect and tease optic tract fibers in poorly fixed human brains. Choosing not to rely on such primitive methodology, most reviewers (Brodal, 1981 ; Walsh & Hoyt, 1969; Williams & Warwick, 1975) comment on the results of experimental animal investigations. However, inferring such pathways from animal models may be dangerous, and one must take into

Fig. 5. Section of human pretectum which contains degenerated axons (arrows) (survival time: one year) between neurons (C) ( x 1000, PPD).

account the interspecies variations in the visual system documented in experimental animal orders (Polyak, 1957).

The mechanism of PPD staining is probably based on paraphenylene-diamine chelating the osmium that has precipitated on lipids, includ- ing by-products of degeneration (Estable-Puig et al., 1965; Sadun & Schaechter, 1985). Oxidized paraphenylene-diamine becomes very opaque and thus is a good marker for lipid elements, such as the myelin sheath of an axon and the remnants of degenerated axons. It has been shown that products of degeneration may per- sist in the primate visual system for many years following injury (Sadun & Schaechter, 1985).

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Ultrastructural examination of serial PPD- stained sections confirmed the identification of pre-terminals and even terminals (ibid.). The PPD method thus permits staining of degener- ated myelinated neural processes even after very long survival periods. This is in contrast to the silver staining methods, which necessarily rely on the dynamic processes of neurofibrillar degenerations that last only about three to seven days following a lesion. The PPD method is therefore superior as a tract tracing method for use in the human visual system.

We have been able to demonstrate projections of the retina to the LGN and to the PT in man with PPD. These pathways are but two of several retinofugal projections identified in various

animals. By means of the PPD technique, a far more complete delineation of the pattern of projections can now be pursued in the human visual system.

ACKNOWLEDGEMENTS

I would like to thank Drs. E. P. Richardson and T. Hedley- White for helping to obtain appropriate human neuropatho- logical specimens. 1 am also grateful to Drs. J. C. Blanks and S. J. Ryan for providing selected brain tissue from mon- keys which had prior enucleations, and to Ms. Ann Dawson for her editorial reviews.

This study was presented in part at the 1981 and 1982 annual meetings of the Association for Research in Vision and Ophthalmology, Sarasota, Florida. This study was sup- ported in part by NEI grants EYO-5894 (AAS) and EYO-3040 (Doheny Eye Foundation electron microscopy core facility).

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