J. Anat. (1985), 141, pp. 139-154 139With 9 figuresPrinted in Great Britain

Axonal and vascular changes following injury to therat's optic nerve

J. A. KIERNANDepartment of Anatomy, The University of Western Ontario,

London, Ontario N6A 5C1, Canada

(Accepted 29 November 1984)

INTRODUCTION

Axons in the optic nerves of birds and mammals do not regenerate after they havebeen severed. In this respect, they resemble the axons of most other tracts of thecentral nervous system and contrast sharply with the optic fibres of fishes andamphibia, which can accurately re-establish their synaptic connections in thebrain.Earlier investigations concerned with the consequences of axotomy in the optic nervehave been reviewed by Cattaneo (1923) and Sperry (1945). In more recent years, theinjured mammalian optic nerve has served as an experimental model in severalstudies of Wallerian degeneration in the central nervous system.The optic axons may be severed by either crushing or cutting the optic nerve. In

the rabbit and rat this operation does not usually result in ischaemic death of theretina, provided that the lesion is more than about one millimetre behind the eyeball:the central retinal artery in these animals enters the optic nerve just behind the opticdisc (Bruns, 1882; Ruskell, 1964). Posteriorly, the optic nerve contains no largeinternal vessels and appears to receive its blood supply through its meningeal cover-ings. Postoperative changes in the retina and optic pathways can therefore beattributed with some confidence to the degeneration of axons. Many of the retinalganglion cells die after their axons have been cut, but many survive (Cattaneo, 1923;Mantz & Klein, 1951; Eayrs, 1952; Muchnick & Hibbard, 1980). Neuroglial cells inthe optic nerve posterior to the lesion become more numerous, and participate inthe phagocytosis of the degenerating axons and their myelin sheaths (Cook &Wisniewski, 1973; Valat, Fulcrand & Marty, 1978). Eventually, the cytoplasmic pro-cesses of astrocytes replace the nerve fibres (Lassmann, Ammerer & Kulnig, 1978;Yeo & Fernando, 1979). At the site of a crush, the optic nerve becomes infiltratedwith collagenous connective tissue (see below). Thus, the histopathological changesare essentially the same as those occurring in other injured tracts of the centralnervous system (reviewed by Windle, 1956; Berry, 1979-; Kiernan, 1979).The optic nerve of the rat is attractive as a test object for experimental manipula-

tions intended to promote axonal regeneration in the central nervous system. Almostall its axons are of the same type and almost all go in the same direction. Con-sequently, any axons seen posterior to the site of a lesion most probably regenerated.A small population of efferent fibres in the rat optic nerve (Itaya, 1980) probablyundergoes retrograde degeneration; no convincing evidence has ever been presentedfor the existence of normal axons in the central stump of the optic nerve aftertransection or after enucleation of the eye (Schlote, 1970). If regeneration should beinduced to occur, its functional efficacy could easily be tested by examining theresponse of the pupil to light. This communication describes the unusually slow

course of degeneration of optic axons in the rat. The effects of the lesion on theblood-brain barrier in the optic nerve are also documented, because vascular perme-ability may be an important factor in determining the success of axonal regeneration(Kiernan, 1978; Kiernan & Contestabile, 1980; Sparrow & Kiernan, 1981). Theresults provide a baseline for the study of therapeutic regimes devised to enhanceregeneration. Three such treatments, all ineffective, were tested in conjunction withthis investigation.

MATERIALS AND METHODS

Operative procedureThe animals used were 52 male albino Wistar rats weighing 150-300 g. For all

operations and intravenous injections they were anaesthetised with intraperitonealpentobarbitone sodium and chloral hydrate, according to the dosage schedule ofValenstein (1961). Postoperatively, the rats were individually caged with free accessto a standard cube diet and drinking water.The left optic nerve was exposed by means of a parasagittal incision 10-15 mm

long whose centre was halfway between the upper eyelid and the midline. The deepfascia medial to the orbital contents was incised close to the skull, immediatelyanterior to and in line with the temporal crest. Dissection was continued, using ablunt instrument (an old scissor blade) in the plane between the skull and theHarderian gland. The most anterior part of the fleshy origin of the temporalismuscle, which encroaches into the orbital region, was scraped away, in an antero-posterior direction. The cone of extraocular muscles was brought into view byfurther blunt dissection between the Harderian gland and the smaller, darker intra-orbital lacrimal gland. The muscles were separated, using fine forceps, to exposethe optic nerve, which was then crushed 2-5 mm behind its point of exit from theeyeball. The crushing instrument was a Hartmann's crocodile-action forceps (DownBros and Mayer & Phelps Ltd, Mitcham, Surrey, England) with jaws 1-5 mm wide.These had been modified by filing away the corrugations and spot-welding into theirplaces pieces of sheet brass to provide two flat, apposable surfaces. This instrumentwas applied at full pressure for approximately 30 seconds. The wound was closed bypushing together the displaced soft tissues and then stitching the skin with four orfive interrupted sutures of braided silk.

In preliminary trials this operative method was found to be superior to approachesthrough the conjunctival sac or through a skin incision posterior to the eye. Thelatter procedures commonly resulted in injury to the duct of the extrorbital lacrimalgland and to the zygomatic branch of the facial nerve, with consequent ulcerationof the cornea.

Experimental treatmentsThe following local treatments were administered at the time of operation.

(a) None (24 rats)A small piece of surgical gelatin sponge (about 1.5 mm3) was placed beside the

crushed optic nerve.

(b) Application of denatured egg albumen (8 rats)A gelatin sponge was soaked in a 10% w/v aqueous solution of egg albumen

(Sigma Chemical Co, St. Louis, Mo, U.S.A.), which is strongly irritant to tissues ofthe rat. The impregnated sponge was placed in ethanol for two hours, then allowed

140 J. A. KIERNAN

to dry, to denature the albumen so that it would not diffuse away from its site ofapplication. A small piece of the dried, albumen-impregnated, gelatin sponge wasplaced next to the crushed optic nerve. It was thought that an inflammation-producing substance might promote axonal regeneration by prolonging the breachof the blood-brain barrier caused by the injury.

(c) Application of trypsin (4 rats)A few granules (approximately 2 mg) of an insolubilised preparation of trypsin

(Enzygel Trypsin-30; Boehringer Mannheim Ltd, Ville St. Laurent, Quebec,Canada) were applied to the site of crushing and left there. This treatment was triedbecause Matinian & Andreasian (1976) claimed that treatment with trypsin promotedaxonal regeneration across sites of transection of the spinal cord.

(d) Intraorbital cholera toxin (6 rats)A small piece of gelatin sponge was soaked in a solution containing 100 ,ug/ml of

cholera enterotoxin in saline (Schwarz-Mann, Orangeburg, NY, U.S.A.) and placednext to the crushed optic nerve. The reasons for using cholera toxin are explained inthe last section of the Discussion.

(e) Intraocular injection of cholera toxin (4 rats)10 ul of a solution containing 50,g/ml of cholera enterotoxin in saline was

injected into the vitreous body of the left eye, through a needle inserted at thecorneoscleral junction. This was done in order to make the toxin available to thesomata of the retinal ganglion cells and perhaps stimulate regeneration of theiraxons. In a preliminary experiment, it had been shown that injection of the samedose of cholera toxin into normal rats' eyes did not cause any histologicallydiscernible change in the retina, though there was some cellular infiltration anddeposition of collagen within the vitreous body.

Administration of tracersThe rats were killed at intervals of 2 days to 34 weeks after crushing the left optic

nerve. Most of the animals were injected intravenously with 1-2 ml of a 10%solution of rhodamine B-conjugated bovine serum albumin (Heinicke & Kiernan,1978), one or two hours before being killed by overdosage with ether vapour. Theywere processed for decalcification and paraffin sectioning, as described below.

In 4 rats, approximately 1 mg of horseradish peroxidase ('Type VI' from SigmaChemical Co, St Louis, Mo, U.S.A.) dissolved in 0-02 ml saline was injected into thevitreous body of the right eye. In these animals, the left optic nerve had been crushed10 weeks previously and no experimental treatments had been administered. Theserats were anaesthetised and perfused with a glutaraldehyde and paraformaldehydesolution 24 hours after the injection of the peroxidase. The optic nerves and chiasma,with the adjacent ventral part of the hypothalamus, were dissected out as a singleblock. Another block consisted of the rostral part of the midbrain, with the superiorcolliculi. Both specimens were processed for the histochemical detection of peroxi-dase by the tetramethylbenzidine method in frozen serial sections. Fixation and laterstages of the technique were carried out exactly as described by Mesulam (1978),incorporating the modifications of Mesulam et al. (1980).

Injury to optic nerve 141

J. A. KIERNAN

Histological examinationThe head of each animal, after removal of the calvaria, the mandible and most of

the skin other than around the left eye, was fixed by immersion in sodium phosphate-buffered (pH 7 2) 4% formaldehyde for 3 days and then decalcified in Clark's (1954)formic acid-sodium formate solution (pH 2-0). Each decalcified specimen wastrimmed to give a block containing both eyes and the anterior half of the brain, forsectioning in the approximately horizontal plane of the optic nerves. The blocks weredehydrated and then double embedded in nitrocellulose and paraffin wax by themethod of Pfuhl, as described by Gabe (1976). Serial sections were cut at a thicknessof 15,um, mounted two to a slide and stained by the following methods: (a) iron-haematoxylin and van Gieson, according to Culling (1974), for general orientationand identification of collagen fibres; (b) a silver method for axons employing physicaldevelopment (Kiernan, 1981, p. 272), counterstained with neutral red; (c) cleared inxylene and mounted without staining, for fluorescence microscopy. The fluorescencemicroscope was a Leitz instrument equipped with a Ploem vertical illuminator todeliver exciting light of wavelength 545 nm (green) and to allow observation ofemitted light of wavelengths greater than 580 nm (orange). This arrangement pro-vides optimal conditions for observation of the fluorescence of rhodamine B-conjugated protein.The sections were examined to determine: (a) survival of retinal ganglion cells:

the number per linear mm of sectioned retina was counted with the aid of a ZeissVideoplan digital image analyser to give an approximate quantitative estimate;(b) position of the lesion; (c) presence or absence of axons in all parts of the opticnerve, anterior to, at, and posterior to the site of the lesion; (d) presence orabsence of extravascular fluorescence due to the injected tracer, in all parts of bothoptic nerves. The normal (right) optic nerve served as an intrinsic control for evalu-ation of the efficacy of axonal staining and for autofluorescence of the tissue.

Electron microscopySix rats, with crushed optic nerves (4 at 10, and 2 at 28 weeks after operation),

none subjected to any experimental treatment, were perfused with Karnovsky's(1965) cacodylate-buffered glutaraldehyde and paraformaldehyde solution. Pieces ofthe left and right optic nerves were then immersed for 3 hours at 22 °C in the samefixative, post-fixed with osmium tetroxide, stained en bloc with saturated aqueousuranyl acetate and embedded in an epoxy resin according to the procedure recom-mended'by Mercer & Birbeck (1972). Semithin sections were stained with alkalinetoluidine blue for optical microscopy. Ultrathin sections were mounted on coppergrids, stained with lead citrate and examined in -an electron microscope.The left optic nerves were examined to seek the presence of both normal and de-

generated axons. The right optic nerve served as a normal control, to ensure thatthe quality of fixation was acceptable.

RESULTS

Changes in the retina after crushing the optic nerve

In 8 rats, the left retina atrophied completely after crushing the optic nerve. Theseanimals had been allowed to survive for 5 days, 5 weeks, 8 weeks, 9 weeks, 10 weeks,14 weeks (2 rats) and 34 weeks. In the remaining 34 rats from which paraffin sections

142

Injury to optic nerve 143

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Fig. 1. Survival of ganglion cells in the retina after crushing the optic nerve. The number in theleft retina is expressed as a percentage of the number in the right (normal) retina of the sameanimal. Animals in which the left retina degenerated are not included. Each symbol representsone animal. 0, Untreated; l, egg albumen applied to optic nerve; *, trypsin applied to opticnerve; A, cholera toxin injected into vitreous body; A, cholera toxin applied to optic nerve.

of the eyes were cut, the retinas survived but the numbers of ganglion cells wereextremely variable (Fig. 1). When ganglion cells were present, there were also axonsin the nerve fibre layer of the retina, these being most numerous near the optic disc.The normal retinas contained 99-6 ± 3.4 ganglion cell nuclei per linear mm of retina(mean ± standard error, in sections 15 ,m thick; no allowance for shrinkage). Inorder to minimise the effects of variation among individual rats, the number ofsurviving ganglion cells in the left retina was, in each case, expressed as a percentageof the number on the right side. The proportion of ganglion cells on the operatedside decreased postoperatively, but the magnitude of the decrease was not related tothe time after crushing the optic nerve. Spearman's ranked correlation coefficient(p) for the data of Figure 1 was not significant (p = 0-274; 0-1 < P < 0-2, from Tablesin Zar, 1984).A small area of extravascular orange fluorescence, due to exudation of the intra-

venously injected labelled albumin, was always present in the optic nerve at the levelof the cribiform lamina, where the axons passed through the sclera (Figs. 2, 3). Thiswas seen on both the normal and the operated sides. Fluorescence in the retina couldnot be critically evaluated owing to appreciable autofluorescence in this region.None of the experimental treatments affected the survival of ganglion cells (Fig. 1)

or the vascular permeability of the initial segment of the optic nerve.

Optic nerve anterior to lesionBetween the optic disc and the site of crushing, the optic nerve was only slightly

narrower than that of the unoperated side. Axons were present at all times, except inthose rats in which no retinal ganglion cells persisted. The number of axons, how-ever, was conspicuously smaller than normal from the second postoperative weekonwards (Figs. 4, 5). Most of the surviving axons ended 0-5-1 *0 mm anterior to the

J. A. KIERNAN

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lesion, but they did not form retraction bulbs. In the first two weeks after crushing,branching axons were often seen anterior to the site of injury, but none were everobserved at later times. From the third to the fourteenth week, many of the axons atthis level turned back and, in favourable sections, they could be traced for up to100 ,am towards the retina (Fig. 6). At 10 and 28 weeks, the electron microscoperevealed only scanty myelinated axons among great numbers of astrocytic processes.

There was never any extravascular fluorescence in the optic nerve anterior to thelesion, except in the connective tissue septa, which were continuous with themeningeal sheath. This appearance was identical to that of the anterior part of thenormal optic nerve.

Site of crushingFor the first two weeks after operation, the site of injury was prominent by virtue

of the presence of numerous erythrocytes within the optic nerve. The lesion waslocated one third to one half of the way between the optic disc and the optic foramen.It was surrounded by loose connective tissue which contained many macrophagesand fibroblasts. At later postoperative times, the optic nerve was surrounded by densecollagenous connective tissue, continuous with its meningeal sheaths (Fig. 7). Thediameter of the optic nerve within this collagenous cuff was greatly reduced, con-stituting about one fifth of normal by 4 to 6 weeks. A few longitudinally alignedcollagen fibres were often present within the optic nerve at the site of crushing fromthe fourth week after operation onwards. The axons in the optic nerve at the site ofcrushing rapidly degenerated into argyrophilic fragments, which persisted for 3-5weeks. At later times, the remains of optic axons were absent from the crushedregion.

Strong extravascular fluorescence, indicative of exudation of the injected tracerprotein, was present within the optic nerve at the site of the lesion for 2 weeks aftercrushing. By the third week, this fluorescence was weaker and it had disappeared bythe fourth week. The connective tissue around the crushed optic nerve displayedmoderately strong fluorescence, of intensity comparable to that of other connectivetissue.

Optic nerve posterior to lesionPosterior to the site of crushing, the optic nerve became progressively narrower

as the postoperative time increased. In the middle and posterior parts of its orbitalcourse, a greatly thickened collagenous sheath surrounded the neural tissue which,by the eighth week, became reduced to about one fifth of its former diameter. Thethick sheath did not extend through the optic foramen, however, and the intracranialportion of the optic nerve shrank to only one third to one half of the diameter of its

Fig. 2. Normal optic disc, showing fluorescence due to intravenously injected rhodamine B-albumin. The brightest fluorescence is in the lumen of the central artery of the retina (arrow)and surrounding connective tissue, and in the choroid (c). A less conspicuous area of vascularpermeability is present within the optic nerve (ON) below and to the right of the central artery.x 80.Fig. 3. The region of vascular permeability at the lamina cribrosa at a higher magnification thanFigure 2, in another normal eye. There are two capillary blood vessels (B V) within the permeablearea, at the right side of the picture. The bright fluorescence in the upper right corner is in thecentral artery of the retina. x 375.Fig. 4. The anterior orbital part of the right (normal) optic nerve, showing argyrophilic axons.x 600.Fig. 5. The left optic nerve of the animal shown in Figure 4. This optic nerve had been crushed21 days previously. Axons are thinner and less numerous than on the normal side. x 600.

Injury to optic nerve 145

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Fig. 6. Argyrophilic axons in the optic nerve anterior to site of crushing, after 65 days. Some ofthe fibres make U-turns back towards the retina, which is above and to the left of the areashown. x 850.Fig. 7. The left orbit, including the site ofcrushing the optic nerve, after 21 days. Collagen showsdarkly with van Gieson's stain. A thick fibrous sheath invests the shrunken nervous tissue of theobliquely sectioned optic nerve (ON). The position of the eye is above and to the right of thepicture.

contralateral counterpart. In about one quarter of the animals, a narrow tubularcavity developed within the optic nerve. This was usually in the posterior orbitalportion, but it occasionally extended intracranially, almost to the chiasma. At earlysurvival times, the cavity contained argyrophilic debris and cells with pyknoticnuclei. From the third week onwards, it was filled with cells of uncertain identity andscanty collagenous fibres.

In the silver stained sections, degenerating axons were easily identifiable as linesof black fragments or as irregularly beaded structures. Normal axons were alsoblack, but were of uniform diameter. By the fifth day after operation, all the axonsposterior to the lesion were degenerating. The axonal debris persisted, though indecreasing amount, until 10-12 weeks after the optic nerve had been crushed. At latertimes, no beaded or fragmented axons were detectable by light microscopy. Fromthe tenth week until the fourteenth week, tortuous axons of normal appearancewere present in silver stained sections of the intracranial portion of the optic nerve(Fig. 8). A few of these were present from 7-9 weeks after crushing, but they weremixed with obviously degenerating fibres. At 14 weeks, when there were no fragmentsof degenerated axons, the only argyrophilic fibres optically visible in the left opticnerve posterior to the lesion were of normal appearance. They were not numerous,there being no more than 10 of them in any single section, and they were most oftenseen 1-2 mm anterior to the optic chiasma. They were never present anterior to the

146

Injury to optic nerve 147

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** * 'Fig. 8. A long argyrophilic fibre resembling a normal axon in the intracranial part of a crushedoptic nerve, 9 weeks postoperatively. x 500.Fig. 9. Optic nerve immediately anterior to the chiasma, 10 we~eks after crushing. The cyto-plasmic processes of astrocytes occupy most of the field and are recognisable by virtue of theirabundant cytoplasmic fibrils. The dark material (arrows) resembles myelin and is presumed to bedegenlerated axonal debris. Electron micrograph. x 8000.

OptiC foramen. The animals in which these axons were present included 4 rats, killed10-14 weeks postoperatively, in which the left retina was absent or contained noganglion cells. The fibres could not, therefore, have been optic axons that had re-generated through the site of crushing. Apparently normal axons were not presentat 21-34 weeks after operation.

Posterior to the lesion, no axons could be found by electron microscopy in theintraorbital part of the optic nerve at the tenth or twenty eighth postoperative week.Connective tissue trabeculae were less conspicuous than they were anteriorly, andthe neural tissue (Fig. 9) consisted almost entirely of astrocytes and their processes.Occasional capillary vessels and oligodendrocytes were also seen. At 10 and 28 weeks,there were still many fragments of myelinated axons lying in the extracellular spaceamong the cytoplasmic processes of glial cells. The degenerated nature of these axonswas evident from the observation that they contained only granular and filamentousmaterial, with no structured organelles such as mitochondria or microtubules. Thesurrounding cells contained inclusions of membranous material, presumably phago-cytosed axonal debris.

With the fluorescence microscope, it was seen that the extravascular fluorescenceat the site of injury extended up to 1 0 mm posterior to the site of crushing for oneweek after operation. From 3-9 weeks, the only extravascular fluorescence behindthe lesion occurred in blood vessels and in small spots in the central cavity, when thiswas present. From 10-14 weeks, a region of weak, patchy, extravascular fluorescencewas present in the optic nerve immediately anterior to the chiasma, extending for-ward about halfway or occasionally all the way to the optic foramen. This fluorescentzone was coextensive in individual animals with the part of the optic nerve containingapparently normal axons in silver stained sections. From 21-34 weeks, there was nofluorescenIce anywhere in the crushed optic nerve except, as previously described, atthe optic disc.

Distribution of horseradish peroxidaseIn sections from the 4 rats in which horseradish peroxidase had been injected into

the normal right eye 10 weeks after crushing the left optic nerve, the distribution ofthe enzyme was examined histochemically in sections 40 ,tm thick. In such sectionsit is easy to identify the terminal fields of projection of fibres that have transportedthe tracer anterogradely: the finely granular blue product of the histochemicalreaction is present in the neuropil. This appearance was seen in the superficial layerof the contralateral superior colliculus, a known site of termination of optic axons,and in another such site, the suprachiasmatic nucleus of the hypothalamus. Therewere light blue fibres in the right optic nerve, and some granular blue material inthe left optic nerve, anterior to the chiasma. The texture did not resemble that seenin the superior colliculus or hypothalamus, however, and there was no convincingevidence of the presence of axons containing peroxidase in the degenerated opticnerve.

Peripheral nervesFrom the third postoperative week onwards, the ciliary nerves, which run along-

side the optic nerve, commonly adhered to the collagenous scar at the site ofcrushing.Small bundles ofaxons entered the thickened sheath of the optic nerve and individualargyrophilic fibres occasionally penetrated deeply into and along the optic nerve,though for no more than 1 mm anterior or posterior to the scarred segment. Theperipheral nature ofsuch fibres was betrayed by their regularly undulating appearancewhich was quite unlike that of normal optic axons. Small peripheral nerves werefound in two of the specimens prepared for electron microscopy. They lay withindelicate trabeculae of connective tissue and the axons were unsheathed by Schwanncells with individual basal laminae.

Effect of treatmentsWhen denatured egg albumen or insolubilised trypsin was placed next to the site

of crushing, the appearances were identical to those in control animals left for thesame lengths of time. The gelatin sponge appeared in the sections as a honeycomb-like material, stained red by the van Gieson mixture. It disappeared after the thirdpostoperative week. The fragments of insolubilised trypsin were strongly stained byneutral red; they persisted indefinitely.

Cholera toxin applied to the site cf crushing provoked an inflammatory reactionwith externally visible swelling in the orbital region for two weeks. At this time therewas heavy infiltration with mononuclear leukocytes around the optic nerve and con-siderable exudation ofrhodamine B-labelled albumin in the connective tissue. Within

148 J. A. KIERNAN

the optic nerve, however, the blood-brain barrier was intact. The toxin may thereforehave suppressed the increase in permeability to the tracer that would otherwise haveoccurred in the blood vessels of the optic nerve. When cholera toxin was injectedinto the vitreous humour in two normal rats, the only abnormality found one weeklater was slight cellular infiltration of the vitreous. Intraocular injection after crushingthe optic nerve resulted in partial detachment of the retina, which assumed a muchfolded configuration and became adherent in places to the lens. The cells of the retinasurvived, and nerve fibres were present in the retina and in the optic nerve anteriorto the lesion. At late survival times (21-34 weeks), retinal ganglion cells were asnumerous following intraocular injection of cholera toxin as in control rats withcrushed optic nerves (Fig. 1). There was no evidence of axonal regeneration into oracross the site of crushing in any of the animals treated with cholera toxin.

DISCUSSION

Degeneration of retinal ganglion cellsRetrograde degeneration of some, but not all of the retinal ganglion cells after

cutting or crushing the optic nerve has been described repeatedly (Cattaneo, 1923;Mantz & Klein, 1951; Eayrs, 1952). In this study there was no significant correlationbetween the postoperative time and the number of surviving neurons (Fig. 1). Com-parable results were obtained by Grafstein & Ingoglia (1982) in a series of 11 mice inwhich the optic nerve had been transected intracranially. The great variability in theretrograde response does not indicate the existence of a special subpopulation ofganglion cells that resist axotomy. Muchnik & Hibbard (1980) have identified suchresistant cells, also recognisable by their dendritic branching patterns, in the retinaof the Japanese quail. In that species, however, almost all the loss of ganglion cellsoccurred during the first two weeks after operation, and there was much less variationamong individual animals than in the present study. If pressure on the optic nerveduring the convalescent period enhanced neuronal degeneration, one would expectthe survival of ganglion cells to have been minimal in those rats in which the orbitaltissues were conspicuously oedematous following local introduction of cholera toxin.This was not the case, however (see Fig. 1). Indeed, none of the experimental treat-ments affected the survival of ganglion cells, and the source of the variation observedin the rat remains unknown.

Reaction at the site of injuryThe most prominent changes at and posterior to the site of crushing the optic nerve

were narrowing of the nervous tissue, encirclement by a thick collagenous sheath andsometimes the formation of a central cavity. The persistent thick sheath may beconsidered to be a hypertrophic dura mater, because thickening of the leptomeningesof the degenerating optic nerve has been shown to last for only 3-5 weeks after injury(Yeo & Fernando, 1979). The central necrosis resembles that seen near sites ofinjury of the spinal cord, and may have been due to local ischaemia (Jellinger, 1976).

Failure of axonal regenerationThe axons of the surviving retinal ganglion cells died back into the anterior part

of the optic nerve, where some of them developed branches or turned back towardsthe retina. Similar changes have been described by others (Ramon y Cajal, 1928,p. 583-596; Richardson, Issa & Shemie, 1982) and interpreted as a transient attempt

Injliry to optic nerve 149

at axonal regeneration. However, in view of the fact that optic axons never enteredthe region of the lesion, it is difficult to avoid the pessimistic conclusion of eankovic(1968) that mammalian optic fibres have no capacity for regeneration. The appear-ances in the rat are not even faintly reminiscent of those seen in fishes (Sperry, 1945;Murray, 1976; Kiernan & Contestabile, 1980) or amphibians (Sperry, 1944; Gaze,1960), in which axons of the optic nerve regenerate successfully.Randomly directed axonal regrowth has been described after placement of small

cuts in the nerve fibre layer of the retina. It has been suggested that the optic axonsmay make more energetic attempts at regrowth there than in the optic nerve (Gold-berg & Frank, 1980; Moulton-Barrett & Berry, 1980; Goldberg, Frank & Krayanek,1983). However, the greatest length of regenerating retinal axons measured byMcConnell & Berry (1982) was only 0 5 mm in 100 days. It is thus possible that thecapability of axonal regeneration in the retina is not greater than in the optic nerve.

Apparently normal axons in silver stained sections of the central stump of thetransected optic nerve were noted by Poscharissky (1909) in the dog and wolf and byRossi (1909) in the rabbit. These authors considered the fibres to be degeneratingaxons that had not become fragmented. The extreme slowness of Wallerian degenera-tion in the mammalian optic nerve has been confirmed by electron microscopy bySchlote (1970), Cook & Wisniewski (1973), Lassman et al. (1978), and in the presentstudy. However, sprouting of undamaged optic axons across the midline has beendetected in young rodents (Lund & Miller, 1975; Hsaio, 1984). The possibility wastherefore considered that some of the long, intact argyrophilic fibres in the intra-cranial part of the crushed optic nerves might be newly formed branches of axonsfrom the contralateral, normal retina. However, branching axons were never seenin the optic chiasma in the silver stained sections, and sprouted axons could not bedetected by anterograde tracing of the intra-axonal transport of horseradish per-oxidase from the normal eye. Another source of intact fibres anterior to the chiasmacould be centrifugal axons, which have been shown to exist in the optic nerve of therat by Itaya (1980). Such fibres would be expected to die back towards the brain withthe passage of time after axotomy in the orbit (Fillenz & Glees, 1961). If some of thefibres observed in the present study were the intact proximal stumps of severedcentrifugal axons, they cannot have been very numerous, because no axons contain-ing normal cytoplasm (indicating continuity with the soma) were seen in the sectionsprepared for electron microscopy.

The blood-optic nerve barrierThe nervous tissue of the normal optic nerve of the rat is not accessible to a

circulating fluorescent tracer, except at the lamina cribrosa. This observation showsthat in the rat the blood vessels of the optic nerve are similar to the equivalent vesselsof the cat, dog, guinea-pig, mouse, rabbit and rhesus monkey (Rodriguez-Peralta,1966; Olsson & Kristensson, 1973). It is possible that the permeation of rhodamineB-labelled albumin into the lamina cribrosa occurs by an indirect route, first intothe loose connective tissue of the orbit and then, by diffusion, through the duralsheath of the optic nerve (Grayson & Laties, 1971). The optic nerve head is the onlysite at which the sheath is permeable (Olsson & Kristensson, 1973). Crushing theoptic nerve caused no change in the size or intensity of the fluorescent region seenat the lamina cribrosa. Probably, therefore, the tissue at that point did not becomecedematous. Baumbach, Cancilla, Hayreh & Hayreh (1978) showed that swelling ofthe optic disc following injury to the optic nerve of the monkey was due not to

150 J. A. KIERNAN

oedema but to axonal enlargement, a presumed consequence of the damming ofaxoplasmic transport.

In mammals, the blood-tissue barrier to plasma proteins is disrupted at the site ofan injury to the brain or spinal cord for 1-20 days; the duration of the failure of thebarrier varies with the size and site of the lesion and with the method of assessment(Lee & Olszewski, 1959; Steinwall & Klatzo, 1965; Mitchell, Weller & Evans, 1979;Noble & Maxwell, 1983). The endoneurial capillaries in the distal stump of a crushedor transected peripheral nerve become permeable for a much longer time, perhapsindefinitely (Mellick & Cavanagh, 1968; Olsson, 1972; Ahmed & Weller, 1979), andan even greater permeability is observed in the presence of actively regeneratingaxons (Sparrow & Kiernan, 1981). The effect of injury on the mammalian blood-optic nerve barrier has not previously been described. The principal change is afailure of the barrier lasting about two weeks and confined to the site of the injury.Thus, the optic nerve behaves in the same way as the brain or spinal cord. A muchless intense fluorescence in the intracranial part of the crushed optic nerve, seen onlybetween the seventh and fourteenth postoperative weeks, is associated with thepresence there of long, unbroken pieces of degenerating axons which lie freely in theextracellular space. The reason for the association is unknown.The changes in vascular permeability in the rat's crushed optic nerve stand in

marked contrast to those previously described in the goldfish, which has been studiedwith the same techniques (Kiernan & Contestabile, 1980). In the fish, a conspicuouszone of vascular permeability accompanies the regenerating optic axons as they growthrough the optic nerve, chiasma and tract and into the tectum of the midbrain. Theassociation of axonal regrowth with vascular permeability is consonant with sug-gestions (Heinicke & Kiernan, 1978; Kiernan, 1978, 1979; Heinicke, 1980) that axonscan regenerate only into extracellular fluid that contains proteins derived from theplasma. A prediction based on this hypothesis is that axons of the mammalian centralnervous system will regenerate if a prolonged failure of the blood-brain barrier ismaintained at the site of axotomy and in the tracts into which the axons are requiredto grow.Although several procedures are known which produce breaches of the blood-

brain barrier lasting a few hours in experimental animals, no method has yet beenfound to cause the vessels to remain uniformly permeable for several weeks (Laursen& Westergaard, 1977; Heinicke, 1982). In the present study, it was not possible tomodify either the blood-optic nerve barrier or axonal regeneration by placinginflammation-producing materials (egg albumen or insolubilised trypsin) in theorbit. Cholera toxin, similarly placed, caused increased permeability of all the bloodvessels in the region except those of the optic nerve. The action of cholera toxin incausing long-lasting oedema is well known (Craig, 1971) but the endothelial cells ofthe central nervous system evidently differ from those elsewhere in being unre-sponsive. Cholera toxin binds to a ganglioside present in the surface membranes ofall eukaryotic cells, and stimulates the enzyme adenylate cyclase to cause increasedsynthesis of cyclic adenosine monophosphate (cAMP) (van Heyningen, 1977). Thenature of the response of the cell to cholera toxin is consequently the same as itsresponse to a hormone or other agent that physiologically increases the productionof cAMP. Berry (1979) has suggested that axonal regeneration occurs in response toneuronal 'growth factors' which may act by stimulating adenylate cyclase. However,the optic axons of the rat could not be induced to regenerate by applying choleratoxin either at the site of transection or to the cell bodies in the retina.

Injury to optic nerve 151

J. A. KIERNAN

SUMMARY

The optic nerve of the rat has been examined by light and electron microscopy,and also for vascular permeability to fluorescently labelled albumin, 2 days to 34weeks after crushing in the orbit. The operation was usually followed by loss of20-70 % of the retinal ganglion cells. Axons could be followed from the retina intothe optic nerve at all postoperative times, but they always ended anterior to thelesion. Evidence of feeble regenerative growth of optic axons was seen in the first fewpostoperative weeks: bifurcating fibres and fibres that turned back towards the eyewere present within the optic nerve anterior to the lesion. At the site of crushing,the optic nerve eventually became a thin cord of astroglia, surrounded and partlyinfiltrated by collagenous connective tissue. Long argyrophilic fibres were con-spicuous in the intracranial part of the crushed optic nerve from the seventh to thefourteenth postoperative week. These were shown by electron microscopy to bedegenerating myelinated axons that had not been phagocytosed. It was conceivablethat axons from the contralateral retina could have sprouted at the chiasma andgrown into the degenerated optic nerve. This possibility was excluded by tracinganterograde axonal transport of horseradish peroxidase injected into the contra-lateral eye.

Intravenously injected fluorescent protein entered the connective tissue of the orbitand the connective tissue trabeculae of the optic nerve, but it did not permeate intothe central nervous tissue except at the lamina cribrosa, where the optic axons piercethe sclera. Permeability at this site was the same on the operated and unoperatedsides. Abnormal permeability of the vasculature was seen at the site of crushing theoptic nerve. Fluorescence there was strongest in the first two postoperative weeksand was not seen after the third week. A much less conspicuous defect of barrierfunction occurred in the intracranial portion of the crushed optic nerve, from theseventh to the fourteenth postoperative week.The responses of the optic nerve of the rat to axotomy contrast markedly with

those of the goldfish, in which the blood vessels become permeable to proteinthroughout the optic pathway and the axons regenerate successfully. Variousattempts were made to increase or prolong the opening of the blood-optic nervebarrier in the rat, in the hope ofenhancing axonal regeneration, but these endeavourswere all unsuccessful.

This work was supported by a grant in aid of research from the Mutiple SclerosisSociety of Canada. I am grateful to Miss Sylvia Davidson, Mrs Sharon Griffin,Mrs Wendy Venturin, Mr John Wijsman and Mr Don Yakobchuk for technicalassistance with histological preparation and electron microscopy, and to Mrs AgnesBentley for typing the manuscript.

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