Regeneration of adult rat CNS axons into peripheral nerve autografts: ultrastructural studies of the early stages of axonal sprouting and regenerative axonal growth.

Journal of Neurocytology 21, 755-787 (1992)

Regeneration of adult rat CNS axons into peripheral nerve autografts: ultrastructural studies of the early stages of axonal sprouting and regenerative axonal growth G. CAMPBELL*, A. R. LIEBERMAN, P. N. ANDERSON, and M. TURMAINE Department of Anatomy and Developmental Biology, University College London, GowerStreet, London WCIE 6BT, UK Received 14 February 1992; revised and accepted 6 July 1992

Summary If one end of a segment of peripheral nerve is inserted into the brain or spinal cord, neuronal perikarya in the vicinity of the graft tip can be labelled with retrogradely transported tracers applied to the distal end of the graft several weeks later, showing that CNS axons can regenerate into and along such grafts. We have used transmission EM to examine some of the cellular responses that underlie this regenerative phenomenon, particularly its early stages. Segments of autologous peroneal or tibial nerve were inserted vertically into the thalamus of anaesthetized adult albino rats. The distal end of the graft was left beneath the scalp. Between five days and two months later the animals were killed and the brains prepared for ultrastructural study. Semi-thin and thin sections through the graft and surrounding brain were examined at two levels 6-7 m m apart in all animals: close to the tip of the graft in the thalamus (proximal graft) and at the top of the cerebral cortex (distal graft). In another series of animals with similar grafts, horseradish peroxidase was applied to the distal end of the graft 2 4 4 8 h before death. Examination by LM of appropriately processed serial coronal sections of the brains from these animals confirmed that up to several hundred neurons were retrogradely labelled in the thalamus, particularly in the thalamic reticular nucleus. Between five and 14 days after grafting, large numbers of tiny (0.05-0.20 ~m diameter) nonmyelinated axonal profiles, considered to be axonal sprouts, were observed by EM within the narrow zone of abnormal thalamic parenchyma bordering the graft. The sprouts were much more numerous (commonly in large fascicles), smoother surfaced, and more rounded than nonmyelinated axons further from the graft or in corresponding areas on the contralateral side of animals with implants or in normal animals. At longer post-graft survival times, the number of such axons in the parenchyma around the graft declined. At five days, some axonal sprouts had entered the junctional zone between the brain and the graft. By eight days there were many sprouts in the junctional zone and some had penetrated the proximal graft to lie between its basal lamina-enclosed columns of Schwann cells, macrophages and myelin debris. Within the brain, sprouts were in contact predominantly with other sprouts but also with all types of glial cell. Within the junctional zone and graft many sprouts showed no consistent, close associations with other cell processes, although some were in contact or adjacent to processes of astrocytes, Schwann cells or macrophages. There was no evidence to suggest that axonal sprouts grew along astrocytic extensions to reach the junctional zone and graft. At eight days many axons in the junctional zone and graft were in contact with Schwann cell processes. Such axons, particularly those in intimate contact with the Schwann cell, were larger than those which had not established contact. By 14 days, most axons in the proximal graft were surrounded by Schwann cell processes, predominantly in basal lamina-enclosed columns. Some axons were associated with astrocyte processes, either in basal lamina-enclosed columns containing only astrocyte processes and axons or in columns containing a mixture of astrocyte and Schwann cell processes. The astrocyte processes involved in such bundles were concentrated at the periphery of the proximal graft, were not seen in the distal graft and probably represent long finger-like extensions of the astrocytes which rapidly form a glia limitans at the interface between brain and graft. This glia limitans was partially constructed at five days, almost complete at 14 days and subsequently became progressively thicker and more complex. At one month the proximal graft had acquired m a n y of the features of a regenerating peripheral nerve and axons were present in large numbers in the distal graft. However the axon-Schwann cell relationships were immature in many of the Schwann cell columns both proximally and distally at one month, and virtually no myelination was apparent. At two months there were numerous myelinated fibres both proximally and distally although there were larger numbers of nonmyelinated axons, many in immature relationship with associated Schwann cells. Thus the graft appears to offer not only support for axonal elongation but also for a substantial degree of maturation of at, least some of the regenerating axons, although (as will be reported elsewhere), the regenerated nerve fibres began to regress after two months. * To whom correspondence should be addressed. 0300-4864/92 $03.00 +.12

9 1992 C h a p m a n and Hall Ltd

756

CAMPBELL, LIEBERMAN, ANDERSON and TURMAINE

Introduction It is n o w clear that injured axons within the adult m a m m a l i a n CNS are capable of extensive regenerative g r o w t h along segments of implanted peripheral nerves (reviewed in Aguayo, 1985; Berry et al., 1986; Bray & A g u a y o 1989; A g u a y o et al., 1990). This has b e e n d e m o n s t r a t e d m o s t clearly by experiments in which CNS n e u r o n a l perikarya are labelled with retrogradely t r a n s p o r t e d tracers applied to the distal portion of a graft several weeks after implantation of the proximal e n d of the graft into the brain or spinal cord (Richardson et al., 1980, 1982, 1984; David & Aguayo, 1981; Benfey & A g u a y o , 1982; Benfey et al., 1985; F r i e d m a n & Aguayo, 1985; So & Aguayo, 1985). Such studies h a v e s h o w n that a variety of n e u r o n a l types in m a n y different parts of the CNS share the capacity for regenerative axonal g r o w t h after injury and have b e e n of great significance in focusing attention on the influence of the local m i c r o e n v i r o n m e n t o n these events. There remain, h o w e v e r , large areas of ignorance and uncertainty about the responses of CNS n e u r o n s to a x o t o m y a n d about the events that follow implantation of grafts into CNS tissue. Ultrastructural observation of grafts of PNS tissue i m p l a n t e d in CNS, for example, h a v e b e e n few in n u m b e r and limited in scope (e.g. W e i n b e r g & Raine, 1980; David & A g u a y o , 1981; Richardson et al., 1982; Chi & Dahl, 1983; Smith & Stevenson, 1988; Hall & Berry, 1989). The p r e s e n t studies w e r e u n d e r t a k e n to clarify the early events in and a r o u n d the proximal e n d s of sciatic nerve autografts i m p l a n t e d into the thalamus of adult rats, particularly those events associated with the sprouting of CNS axons, the invasion of the graft b y such sprouts and their s u b s e q u e n t elongation a n d maturation within the grafts. Parts of this w o r k h a v e b e e n p u b l i s h e d in abstract form (Campbell et al., 1989a,b, 1990). Materials and methods

Animals All experiments involved Sprague-Dawley albino rats, bred in our animal unit or purchased from Charles River. All of the rats were adult and 180 g or more in body weight at the time of graft implantation.

Graft implantation Animals were anaesthetised with ether and then injected intraperitoneally with Sagatal (0.5 ml kg-1; M & B, 60 mg in 1 ml). Prior to anaesthesia the animals were given a subcutaneous injection of 0.1 ml of atropine (Antigen Ltd, 600 ~g in 1 ml). The skin was incised parallel and caudal to the femur in the left thigh and the sciatic nerve exposed by separating overlying muscles and stripping away their fascia. The two major branches of the sciatic nerve were separated by peeling away the epineurium and a segment of

la

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Distal graft

C

Proximal graft

i

TRN

.s t i ~.

C :;i; [

Parenchymal

_ j

border zone

9 "...:.:.::.'~;~..:.. ~

..... "+:,i.

ions / Parenchym&:-~':..:' '-"':~{'"

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Junctionalzone of graft

Fig. 1. Schematic drawings illustrating the site and route of graft implantation into the thalamus and the approach used to select tissue for subsequent EM analysis. (a) Coronal section of the rat brain at mid-rostrocaudal level of the thalamic reticular nucleus (TRN). A peripheral nerve graft inserted through the skull and meninges (not shown), the cerebral cortex (C) and the hippocampus (H), ends in the region of the ventrobasal nucleus of the thalamus. Levels of the graft defined as proximal and distal are indicated. (b) Portion of an osmicated, 200 ~m Vibroslice section cut in the horizontal plane through the thalamus at the level of the proximal graft. The trapezium outlines the area for EM analysis, which includes a transverse slice of the graft plus adjacent parenchyma of the ventrobasal nucleus (VB). IC, internal capsule. (c) Semithin or ultrathin section of the block removed from the Vibroslice section in (b) indicating the zones and features of the graft/brain interface. common peroneal nerve or tibial nerve 1.5-2.0 cm long was removed and placed in synthetic culture medium (Eagles MEM, IX with Earle's salts, withotft L-glutamine) (Gibco). The nerve was prepared for grafting by trimming away smaller nerve branches and any residual epineurial components such as fat and large blood vessels. The epineurium normally retracted some 1-2 mm from the nerve tips. Using a 10/0 Ethilon suture with a 4.75 mm curved needle (Ethicon) a length of the thread with needle attached and long enough to allow the nerve to be sutured to the dura, was anchored to the centre of the nerve segment by a knot tied through the

CNS axon regeneration into PNS grafts perineurial sheath. A square piece of parietal bone (ca 3 x 3 mm) was removed from the cranium to expose the cortical surface. The graft was then sutured to the dura and at a position estimated to be directly above the dorsal thalamus (using stereotaxic coordinates of Pellegrino and colleagues (1979) and measuring from bregma) a small slit was made in the dura. Using the fine end of a drawn-out Pasteur pipette one end of the graft was pushed through the hole in the dura into the brain to a depth of 6-8 m m (Fig. la). In some cases the glass pipette was pushed several times into the brain before the graft was inserted. The distal end of the graft protruding from the brain was not ligated. In some animals it was tucked under the temporal muscle. The scalp was then sutured and the animal allowed to recover under a warm lamp. Some of the animals used only for LM studies had a small amourit of the cerebral cortex ablated either by suction or by scalpel prior to implanting the graft.

Retrograde labelling experiments Fourteen animals were used for this experiment. After postgrafting survival periods of 1 month to 4.5 months animals were anaesthetised with ether and Sagatal (see above). The distal free portion of the graft was located and identified by its attached black suture and was fully exposed by removing the thickened perisoteum. The graft was cut across about 2 m m from its distal extremity and crystals of horseradish peroxidase (HRP, Sigma type VI) were jabbed into the cut end with a sharp needle. Small pads of gelfoarn previously soaked in 25-50% HRP (in DMSO) and dried were then pressed close to the cut tip of the graft beneath the thin tight periosteum. In this way the gelfoam pad was anchored and held close to the cut end of the distal graft. In all cases the distal graft tip was then covered with Vaseline and the scalp sutured. The animals were killed 24-48 h later. Further details are given in Morrow and colleagues (1993).

Tissue processing for light microscopy Animals were anaesthetised with ether and given an overdose of Sagatal (1.0-1.5 mlkg-l; i.p. and then perfused through the left cardiac ventricle with 100-200 ml of 0.1 M phosphate buffer followed by 300-500 ml of 3% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 at a pressure of 80 m m of mercury. Brains were carefully removed leaving the graft intact and placed overnight at 4 ~C in 0.1 M phosphate buffer containing 10% sucrose. The brain was trimmed to a block which included the rostral extent of the thalamic reticular nucleus and the caudal extremity of the superior colliculus and was immersed in Tissue-Tek OCT compound (Miles Laboratories) and frozen in liquid nitrogen. The brain was then sectioned in the coronal plane at 40 ~m on a cryostat. The sections were left to dry at room temperature on subbed slides for 3-4 h and were then reacted with tetramethyl benzidine (TMB) according to the method of Mesulam (1978) using 2 ml of 0.3% I-I202 per 100 ml of reaction medium. After drying overnight the slides were counterstained with Neutral Red, rapidly dehydrated through ethanols, cleared in Histoclear (National Diagnostics) and coverslipped. For further details see Morrow and colleagues (1993).

Tissue processing for electron microscopy Animals were anaesthetised as described above and then

757 perfused through the left cardiac ventricle with 500 ml of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M Millonig's phosphate buffer (pH 7.4) at a pressure of 80-100 mm of mercury. The brain and graft were then removed from the skull and placed in fresh fixative overnight at 4~C. Horizontal (200 Ixm) or coronal (100 ixm) sections were cut on a Vibroslice (Campden Instruments) and collected in serial order in 0.1 M phosphate buffer. After being osmicated (1 h in 1% OsO4 with 0.1 M phosphate buffer) the sections were stained for 45 min in 2% uranyl acetate in sodium acetate buffer at 4~C, dehydrated in ethanols, cleared in propylene oxide and embedded in Araldite between two slides coated with Releasil A (Down Coming) mould releasing agent (Fig. lb). Semi-thin (1 ixm) sections were cut with glass knives and stained with Toluidine Blue (Fig. 2) adjacent to thin sections cut with a diamond knife on a Reichert Ultracut ultramicrotome. Thin sections (Fig. lc) were collected on mesh grids and single slot grids coated with a thin formvar film, counterstained with lead citrate and viewed in a Philips 300 electron microscope.

Data analysis Sections prepared for analysis by LM were systematically scanned under brightfield illumination. The positions of labelled cells were recorded in camera lucida drawings of the sections. For the EM study both semi-thin and thin sections were cut at both proximal and distal levels of the graft in every animal. The proximal level was in the thalamus, within 0.5 mm of the tip of the graft. To ensure consistency between animals the distal graft was examined in all animals at the level of the superficial cerebral cortex, usually 6-7 mm from the proximal tip of the graft. The semi-thin sections were drawn and locations of all EM photographs were marked on the drawings. Thin sections were systematically scanned and photographed at a range of magnifications. Low power (scan magnification) photographic montages were made of the sections on single slot grids to facilitate the localisation of higher magnification photographs.

Results OBSERVATIONS BY LIGHT MICROSCOPY AFTER RETROGRADE LABELLING WITH HRP The L M - H R P findings are r e p o r t e d in detail e l s e w h e r e ( M o r r o w et al., 1991, 1993). H e r e w e s u m m a r i z e briefly only the principal findings relevant to the interpretation of the EM studies. The L M - H R P results derive f r o m f o u r t e e n rats of b o t h sexes w e i g h i n g b e t w e e n 180 g a n d 500 g at the time of graft i m p l a n t a t i o n a n d surviving for 24 d a y s to 4 m o n t h s before r e t r o g r a d e labelling followed b y sacrifice. The n u m b e r of labelled cell bodies varied b e t w e e n 0 a n d 750. Both c o m m o n p e r o n e a l a n d tibial n e r v e grafts p r o d u c e d labelled cells within dorsal thalamic nuclei, m o s t l y within 200 txm of the graft tip. T h e s e labelled cells all d i s p l a y e d the m o r p h o l o g i c a l characteristics of thalamocortical projection cells. In addition, w i t h tibial n e r v e grafts there w e r e large n u m b e r s of labelled cells w i t h i n the thalamic reticular nucleus (TRN). The m a r k e d p r o p e n s i t y

759

CNS axon regeneration into PNS grafts for neurons of the TRN to regenerate into sciatic nerve grafts confirms the previous findings of Benfey and colleagues (1985) and will be described in greater detail a n d discussed elsewhere. For the present paper the important point is that adequate n u m b e r s of CNS n e u r o n s send regenerating axons into grafts placed in the thalamus a n d such grafts are therefore suitable for studies of the ultrastructure of CNS axons and their e n v i r o n m e n t in the course of regenerative growth into PNS grafts. OVERVIEW OF THE MATERIAL STUDIED BY EM The findings reported here derive from a selected group of animals d r a w n from a m u c h larger group of grafted animals, the others of which were either not analysed in detail because of poor fixation or inappropriate graft tip localization or because t h e y form part of a subgroup of long term survival animals (more than two months) which will be described elsewhere. Most of the observations and all of the illustrations in this paper are based on eight animals surviving from five days to two m o n t h s after graft implantation. In all, the graft passed almost vertically t h r o u g h the cerebral cortex a n d hippocampal formation a n d into the dorsal thalamus (Fig. la; see Table I for further details). TERMINOLOGY We recognize the following regions of graft-brain interface (see Figs lc, 2,3): (i) the graft proper (G, Fig. 2a-d); (ii) the junctional zone of the graft (JZ, Fig. 2a,c); (iii) the p a r e n c h y m a l border zone (PBZ, Figs 2a-d); (iv) normal CNS parenchyma. The CNS p a r e n c h y m a a r o u n d the parenchymal border zone displays no or very few abnormalities at any post-graft survivai

Table 1. Details of animals, grafts and graft position.

Animal number GC66 GC56 GC69 GC63 GC70 GC60 GC61 GC84

Weight (g) Graft type

Post-graft survival time

Terminus of proximal graft

205 240 220 200 200 240 240 180

5 days 8 days 8 days 14 days 14 days i month 2 months 2 months

LPN LPN VB VB VB LGN LPN APN/LPN

Peroneal Peroneal Peroneal Peroneal Peroneal Peroneal Peroneal Tibial

LPN: lateral posterior nucleus, VB: ventrobasal nucleus, LGN: lateral geniculate nucleus, APN: anterior pretectal nucleus.

period a n d merges w i t h o u t distinct boundaries into the parencyhmal border zone which contains degenerating and reactive axons, axon terminals and dendrites, reactive glial cells, and particularly in the first few days after grafting, large n u m b e r s of axonal sprouts. The thickness and distinctiveness of the parenchymal border zone are variable, but even in the short term survival animals (5-14 days), in which it is thickest a n d most abnormal, it extends no more than about 100-150 ixm from the interface b e t w e e n the brain and the graft. At the earliest survival times this interface is marked by a forming glia limitans and from 14 days o n w a r d s by an almost continuous glia limitans, which separates the parenchymal border zone from the junctional zone of the graft. The junctional zone is of variable thickness and distinctness and of

Fig. 2. Semithin (1.0 jxm), Toluidine Blue-stained, transverse sections of the proximal (a,c) and distal (b,d) graft (G) and surrounding brain parenchymal border zone (PBZ) five days after grafting (a,b) and one month after grafting (c,d). The grafts consist of the remnants of the original nerve fibres, of which degenerate myelin is the most striking component in (a), (b) and (c). In (d) (distal graft at one month) most of the myelin debris has been cleared and columns of Schwann cells containing nonmyelinated axons predominate. The graft at distal levels (b,d) is clearly demarcated from the parenchymal border zone by a thin, multilayered perineurium and a vascular and collagenous epineurium. In contrast, there is neither epineurial nor perineurial tissue at proximal levels of the grafts ((a) and (c)) where components of the graft are separated from the parenchymal border zone by a junctional zone (JZ) which is clearly identifiable at five days but indistinct at one month. Two small branches (B) of the peroneal nerve graft are seen within the epineurium in (b). Note the difference in the quantity and density of degenerating myelin and debris-laden macrophages between the five day and one month grafts and between proximal and distal graft levels. The one month graft at proximal level (c) contains several large accumulations of collagen. Scale bar = 100 ~m and applies to entire Figure. Figs 3-33. Electron micrographs of transverse sections through the proximal graft and adjacent parenchymal border zone between five days and one month after graft implantation. Fig. 3. Five days after grafting. Low power electron micrograph of the interface between graft and brain (this interface is also shown in a semi-thin section in Fig. 2a). The graft (G)~ only the most peripheral part of which is seen, is dominated by degenerate nerve fibres from which axons have disappeared and which contain myelin debris, macrophages and Schwann cells. In the endoneurium around these basal lamina enclosed tubes are collagen fibres, macrophages, fibroblasts and their processes and empty basal lamina tubes. The parenchymal border zone (PBZ) contains large numbers of myelinated fibres, most of them normal in appearance, and many astrocytes and oligodendrocytes. A few abnormal (reactive) axons, one of which is marked with an asterisk (*) are aIso apparent. Between th'r parenchymal border zone and graft tissue is a poorly delineated junctional zone (JZ) containing macrophages, fibroblasts, a few Schwann cells, and processes of uncertain origin, collagen and empty basal lamina tubes. Scale bar = 10 ~m.

760

CAMPBELL, LIEBERMAN, A N D E R S O N and T U R M A I N E

Table 2. S u m m a r y of the principal ultrastructural findings. Parenchymal border zone Surv. time

5d and 8d

14d

Proximal

Fig. nos Distal

GL thin and incomplete.

3 4a 8

Numerous axonal sprouts; many in bundles some contact AS, OL, M.

4 5 6 8

Junctional zone Fig. nos Proximal

GL incomplete. 34

Some sprout- 34 like profiles, present, but fewer than proximally; some associated with AS, OL, M.

GL thicker and almost complete,

GL virtually complete,

-

Sprouts as at 5-8 days

13

Sprouts as at 5-8 days

GL complete and multilayered

17a GL complete 19 and 20 multilayered,

Fewer sprouts; arrangement and relations similar to 5-8 days

17a Some sprout18 like profiles 19 still present, 20

GL complex and multilayered.

26 27 28

Similar to one month except GL thicker,

41

Few sprouts present.

27 28

Very few sprouts.

-

-

lm

2m

Small axonal sprouts, fine processes of SC, AS, M all present, but limited intimacy between sprouts and other cell processes.

Graft Fig. nos Distal

Fig. nos Proximal

Fig. nos Distal

3 7 8

Similar to proximal but fewer sprouts or other cell processes

-

All PNS axons degenerate.

2a 3

Occasional large bundles of sprouts,

34

A few axonal sprouts, most lacking contacts with other cell processes

9 10 11 12

Similar to 5--8 days but narrower.

-

More sprouts 14 present than at 15 five days some 16 enlarged and invaginated into SC, and some in contact with AS in SC columns.

37 40

Most axons in columns of SC and/or AS processes

Similar to 5-8 days; fibroblast processes also present. Many sprouts in contact with SC processes, some intimately.

37 Indistinct and 17a Heavily 40a contains 21a collagenous mixture of SC, and similar to AS and OL peripheral processes, nerve Constituents of epineurium. graft appear to abut on GL. AS 'bridges' present. Similar to one month.

26

As at 1 month; 41 contains some 42 nerve fibres and AS 'bridges'.

Some small axons only loosely associated with SC and AS. Similar to one month. More axons, many myelinated by SC.

Fig. nos

Axonal sprouts between layers of perineurium but not beneath perineurium.

SC columns 35 mostly 36 anaxonal; few putative axons.

17 23 24 25

Many axons 38 present, most 39 associated with SC. Intimate associations 17b fewer than in 17c proximal graft. 22

29 30 31 32 33

Similar to proximal graft but no AS present.

41 42 43 44

Some small 29 axons loosely 30 associated with SC and AS. Abbreviations:AS, astrocyte; GL, glia limitans; M, microglial cell; OL, oligodendrocyte; SC, Schwann cell.

c h a n g i n g c o m p o s i t i o n w i t h t i m e . It is fairly s h a r p l y d e m a r c a t e d f r o m t h e S c h w a n n cell c o l u m n s a n d d e g e n e r a t e m y e l i n of t h e graft p r o p e r at s h o r t p o s t graft s u r v i v a l t i m e s (Fig. 3) b u t is h a r d e r to d e f i n e later o n (at l e a s t i n t h e p r o x i m a l graft), as c o m p o n e n t s of t h e graft p r o p e r c o m e to lie close to t h e glia l i m i t a n s .

OBSERVATIONS BY ELECTRON MICROSCOPY: PROXIMAL GRAFT I n this a n d t h e f o l l o w i n g s e c t i o n w e d e s c r i b e E M o b s e r v a t i o n s o n t h e p r o x i m a l graft a n d its e n v i r o n s w i t h i n 0.5 m m of t h e graft tip (Figs 3-33) a n d t h e distal graft a n d its e n v i r o n s 6 - 7 m m f r o m t h e tip (Figs 34 44).

Fig. 4. Five days after grafting. (a) Survey electron micrograph of parenchymal border zone. Abutting the graft at top right is an immature and incomplete glia limitans (arrowheads). Just below the glia limitans are several glial cells including a m e d i u m oligodendrocyte (OL) a n d n u m e r o u s astrocyte processes (a). Several clusters of small n o n m y e l i n a t e d axons are apparent in this region (large arrows), as are several reactive or degenerating axons and dendrites (*). (b,c) Enlargements of arrowed areas b and c, showing groups of small n o n m y e l i n a t e d axonal profiles (small arrows). These profiles lie a m o n g astrocyte processes (a) and fine finger-like extensions of such processes beneath the glia limitans. Scale bars: (a) 2 ~m, (b,c) 0.5 ~m.

762 In both cases the figures are arranged in order of post-grafting survival times but are described in terms of the evolution of change in (i) the parenchymal border zone, (ii) the junctional zone, (iii) the graft. Summaries of the main changes are contained in Table 2 and further details are provided in figure legends.

The parenchymaI border zone Glia limitans. The parenchymal border zone (PBZ, Fig. 3) varied in width from 100-150 txm at 5-8 days postgrafting to 40-60 Ixm at two months. It was demarcated from the junctional zone of the graft (JZ, Fig. 3) by astrocyte processes forming a glia limitans which at five days was incomplete and with a discontinuous basal lamina, at 14 days was almost continuous and at two m o n t h s was multilayered, complex a n d often t h r o w n into deep folds extending both into and away from the PBZ (Figs 26-28). Where the glia limitans was incomplete, especially at 5-14 days, clusters of axonal sprouts (see below) (Fig. 8) and a m o n g them, occasionally, astrocyte processes, appeared to be passing from the PBZ into the junctional zone. At 14 days, and more so at one m o n t h it was possible to trace finger-like bundles of astrocyte processes, accompanied by basal lamina, from the glia limitans deep into the junctional zone (Fig. 17a). Such glial protrusions or 'bridges' (Weinberg & Raine, 1980) would appear in other planes of section as fibre-like astrocytic columns. Some of these astrocyte bundles were associated with axon sprouts (see below).


Neuropil, degenerating and reactive neurites; glial cells. Deep to the glia limitans the neuropil of the parenchymal border zone was characterized by the presence, scattered within areas of otherwise normal neuropil, of large n u m b e r s (at 5-8 days) to very few (at two months) abnormal axons and dendrites. Some, with electron dense cytoplasm were evidently degenerate; others were swollen and/or packed w i t h abnormal n u m b e r s of organelles (asterisks, Figs 3, 4a). The abnormal neurites were scattered within a neuropil of otherwise normal appearance. Astrocyte, microglial a n d oligodendrocyte cell bodies and processes were all apparent (Figs 3, 4a, 6, 13) and m a n y s h o w e d reactions to graft insertion: reactive astrocytes a n d cells with the ultrastructural characteristics of m e d i u m or light oligodendrocytes (Figs 4a, 13) which are rare in normal adult CNS tissue (Mori & Leblond, 1970; Parnavelas et al., 1983) were c o m m o n at 5-8 days postgrafting and present in small numbers at later survival times. At one and two m o n t h s some of the myelinated axons h a d thinner sheaths t h a n myelinated axons deeper within the parenchyma, indicating recent myelination. This was especially obvious at one m o n t h postgrafting (Figs 18-20).

Axonal sprouts. T h r o u g h o u t this zone, but most conspicuously in the superficial region close to the glia limitans, were large n u m b e r s of small n o n m y e l i n a t e d axon-like profiles, most b e t w e e n 0.05 txm and 0.2 ~ m in diameter (Figs 4b, c, 5a,b, 6, 13, 18-20, 27, 28). They were present in large n u m b e r s at five and eight days,

Fig. 5. Five days after grafting. A large aggregation of fine nonmyelinated putative axonal sprouts. These sprouts, shown at low magnification in (a) and some at higher magnification in (b), lie beneath an astrocyte process (a) adjacent to the endothelium (e) of a blood vessel and very close to the brain/graft interface. Close by are myelinated fibres of normal appearance. Most of the sprouts contain microtubules and many also contain a single neurofilament and a vesicle or tubule of smooth endoplasmic reticulum. Note the smooth and generally circular outline of the sprouts and the almost crystalline way they are packed together. Scale bars: (a) 1.0 txm, (b) 0.5 Ixm. Fig. 6. Eight days after grafting. Small axonal sprouts and some larger vesicle-containing axonal profiles closely apposed to the soma and processes of cells with the ultrastructural characteristics of microglia (M) within the parenchymal border zone close to the graft interface. Scale bar = 0.5 Ixm. Fig. 7. Eight days after grafting. The junctional zone, packed with fine glial processes, collagen, microfibrils, a few putative axonal sprouts (arrows) and degeneration debris, lying between the basal lamina-covered astrocyte processes (a) of the forming glia limitans (arrowheads) at the left and (at right) part of a basal lamina-enclosed tube, containing myelin debris engulfed by a macrophage (M), which marks the most peripheral portion of the graft. Scale bar = 0.5 ~m. Fig. 8. Eight days after grafting. A different region of the junctional zone. In this region, the brain/graft interface has an incomplete glia limitans. A thick, tortuous astrocyte process (a) which is onJy partially covered with basal lamina (arrowheads) appears not to appose other astrocyte processes a~ the surface of the parenchymal border zone (bottom of figure) and the basal lamina appears to end at the open arrow. Putative axonal sprouts (some arrowed) are present both in the junctional zone (right of figure), and in the parenchymal border zone (left) below the glia limitans. Long, thin, moderately electron dense cell processes on the right emerge from a macrophage cell body (not shown). Scale bar = 1.0 txm. Figs 9-11. Eight days after grafting. Axonal sprouts (some arrowed) in the peripheral portion of the graft. The axons are adjacent to degenerate myelin (Fig. 9) proteinaceous extracellular material (Figs 9-11) and cell processes which cannot be identified with certainty but may be of both Schwann cell and macrophage origin. Scale bars: (Fig. 9) 0.5 ~m (applies also to Fig. 10); (Fig. 11) 0.25 ixm. Fig. 12. Eight days after grafting. A large Schwann cell process (S), surrounded by a loose basal lamina, is associated with several axons (e.g. at arrows) impressed into its surface. Such close associations between axonal sprouts and Schwann cells are comparatively rare within the proximal graft at this survival time. Scale bar = 0.5 Ixm.

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were less n u m e r o u s at 14 days, and infrequent at one and two months. These axon-like processes had a r o u n d e d , smooth contour, a pale cytoplasmic matrix devoid of organelles in a few cases, but in most cases containing a small n u m b e r of microtubules (usually 2-5; Fig. 5b). In some, small saccules of smooth endoplasmic reticulum, neurofilaments, a single small mitochondrion, lysosome-like dense bodies, dense core vesicles or synaptic-like vesicles were also apparent (Figs 5b, 6, 19, 20, 27, 28). In some of the smallest profiles only a single neurofilament or microtubule could be detected. M a n y of these processes were tightly packed together to form small bundles within which the individual profiles sometimes appeared to be arranged with almost crystalline regularity (Fig: 5a,b). Smaller or larger bundles of these axon-like profiles were often partially enclosed by narrow astrocyte processes or were impressed into the surface of larger astrocyte processes. Such groupings were particularly apparent just below or within the glia limitans (Figs 4, 19, 20, 27, 28), a n d were often present in regions where the glia limitans was incomplete (Fig. 8). They were also c o m m o n l y concentrated immediately adjacent to blood vessels (Fig. 5a). They

were also observed in contact with, and sometimes impressed into the surface of, myelin sheaths and oligodendrocytes (Fig. 13), and the cell bodies a n d processes of microgliaI cells (Fig. 6). Such relationships with glial cells and with myelin sheaths were observed at all survival times. On the basis of their axon-like ultrastructural characteristics, their location within the region of tissue in which axons were d a m a g e d at the time of graft implantation, their large numbers, their size and their fate (see later) we identify these axon-like nonmyelinated profiles as newly formed, regenerating axonal processes and will refer to t h e m as axonal sprouts. The validity of this conclusion will be considered in the Discussion. The junctional zone

The junctional zone was well defined at eight and 14 days and less so at five days a n d 1-2 months. At five and eight days it consisted of an extensive extracellular space within which were present collagen fibres, a few putative axonal sprouts, macrophages and considerable n u m b e r s of narrow finger-like or sheet-like cell processes, which appeared" to be heterogeneous in

Figs 13-16. Fourteen days after grafting. Fig. 13. A cell with the ultrastructural characteristics of a light/medium oligodendrocyte (OL) in the parenchymal border zone adjacent to the graft. Small clusters of sprout-like axons (arrows) are impressed into the oligodendrocyte plasmamembrane in several places. Scale bar = 1.0 ~m. Fig. 14. Small axons (some arrowed) non-intimately associated with Schwann cell (S) and astrocyte (a) processes, enwrapped in a loose basal lamina (arrowhead) and partially surrounded by thin fibroblast processes (f) within the graft. Scale bar = 0.5 ~m. Fig. 15. Moderate-sized axons (arrows), much larger than those in Fig. 14, enwrapped by Schwann cell processes (S) in a basal lamina-enclosed column in the graft. Scale bar = 0.5 ~m. Fig. 16. Mixed column of intermediate filament-rich astrocyte processes (a) and Schwann cell processes (S) associated with axons (some arrowed). The column is enclosed by basal lamina and in places a second basal lamina is apparent (arrowheads). Scale bar = 0.5 ~m. Figs 17-20. One month after grafting. Fig. 17. Survey electron micrograph of the interface between the parenchymal border zone (upper left) and the graft. A clear junctional zone cannot be identified in this region (the same interface is shown in a semithin section in Fig. 2c). This region of parenchymal border zone consists mainly of astrocyte processes (a) some of which enclose small clusters of axonal sprouts (solid arrows). Other astrocyte processes appear to be part of a complex glia limitans which protrudes into the graft (between arrowheads) and probably extends into the transversely sectioned columns of intermediate filament-rich astrocyte processes, some of which are labelled (a) and mixed astrocyte and Schwann cell processes (some Schwann cell nuclei are labelled, S). The graft is largely made up of these basal lamina-enwrapped columns. The columns are widely spaced in an endoneurial matrix containing collagen, macrophages and fibroblasts (f) the processes of which show a tendency to compartmentalise the columns. Many columns are associated with axons, for example columns b and c, which are shown at higher magnification in Fig. 17b and c, where the small axons (some arro~ved) are seen partially enwrapped by astrocyte processes (a) and Schwann cells and their processes (S). Column b appears to be constituted predominantly by astrocyte processes, whereas c appears to contain a mixture of astrocyte and Schwann cell processes. Scale bars: (a) 2.0 ~m, (b,c) 0.5 ~m. Fig. 18. Small oligodendrocyte processes (*) associated with a thinly myelinated axon just beneath an astrocyte process (a) of the glia limitans. The similarity in size and organelle content between the tongues of oligodendrocyte cytoplasm and putative axonal sprouts (arrows) is clear. Scale bar = 0.25 ~m. Figs 19, 20. Pockets of small to moderately-sized putative axonal sprouts (some arrowed) and oligodendrocyte-myelinated axons, tightly surrounded (Fig. 19) or loosely encased (Fig. 20) by astrocyte processes (a). Many of the astrocyte processes contribute towards the glia limitans (arrowheads mark the basal lamina of the glia limitans). Scale bar = 1.0 ~m (applies also to Fig. 19).

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terms of their ultrastructural characteristics, and which were more numerous at eight days than at five days (Figs 7, 8). Most of these processes were of irregular shape, of moderate electron density and contained variable amounts of filamentous and floccular material and organelles. Many resembled macrophage, astrocyte or Schwann cell processes and some were traced into continuity with larger processes more readily identifiable as such. The few axon sprouts present were small round, electron lucent (arrows, Figs 7, 8) and similar to those of the parenchymal border zone. At 14 days the junctional zone was similar but fibroblast processes were now common. At one and two months the junctional zone was indistinct because graft components abutted directly onto the glia limitans (Fig. 17a), but was a complex meshwork of interdigitated CNS and PNS elements - astrocyte protrusions from the glia limitans, Schwann cellenwrapped nonmyelinated and myelinated axons, collagen, fibroblasts and macrophages (Figs 17a, 21a, 26). Because of this intermixing of constituents of the graft it was difficult to determine whether the few basal lamina-enwrapped Schwann cell processes that appeared to be below the glia limitans were within the CNS parenchyma or were part of the junctional zone. In one region of the one month animal where the glia limitans appeared to be incomplete a few oligodendrocyte myelinated axons had penetrated into the graft. Some of these were enclosed by astrocyte processes (Fig. 21).

The graft General appearance. The graft at 5-8 days consisted of basal lamina-enwrapped Schwann cell columns (bands of B~ngner), many containing axonal and myelin debris and macrophages, and a matrix of fibroblasts and collagen. No intact nerve fibres were present: even three days after graft insertion most nerve fibres were recognizably undergoing degeneration (unpublished observations). In the longer survival animals regenerating axons were also a feature of these columns. Surrounding the columns were numerous fibroblasts, macrophages, empty basal lamina

tubes and some Schwann cells in an extracellular matrix containing collagen and microfibrils (Figs 3, 17a). There was considerably more debris and macrophages at five days than at two months (compare Figs 2a and 3 with Figs 2c and 17a). In the one and two month animals many of these columns also contained and in some cases were dominated by astrocyte processes (Fig. 17a).

Schwann cell columns. These columns contained both cell bodies and processes of Schwann cells. At all ages the Schwann cell processes were either medium-sized and apposed one another via large expanses of membrane and therefore resembled Schwann cells of the normal peripheral nerve (Figs 15, 25) or were small, highly irregular in shape and apposed at a few small contact points (Figs 14, 24). These small processes had the appearance of Schwann cell processes within a denervated peripheral nerve trunk (Weinberg & Spencer, 1978). Schwann cell processes were of variable overall electron density and contained differing numbers of organelles including loosely packed intermediate filaments and, most characteristically, small dropshaped elements of smooth endoplasmic reticulum and other short saccules of smooth endoplasmic reticulum in continuity with short saccules of rough endoplasmic reticulum (Figs 12, 16, 25). The columns of Schwann cells tended to be separated from one another and from macrophages in the connective tissue space by long, thin fibroblast processes (Fig. 14) which at one and two months often totally circumscribed the columns, delineating 'minifascicles' and probably representing future perineurium (Ahmed & Weller, 1979; Anderson et al., 1983) (Figs 17a, 30, 33). The columns were sometimes encased by a double basal lamina, the outer of which was only loosely associated with the inner one. Some Schwann cell processes enwrapped other Schwann cell processes. Astrocyte processes. Astrocyte processes were first present in the graft in small numbers at 14 days where they predominated at the periphery of the graft and either formed their own basal lamina-enwrapped columns with a few axons or formed mixed columns of

Figs 21-24. One month after grafting. Fig. 21. Low power (a) and high power (b) electron micrographs of an ofigodendrocyte-myelinatedaxon encased in a bundle of transversely sectioned, intermediate filament-rich astrocyte processes (a') The bundle is enclosed by a basal lamina and surrounded by fibroblast processes. It lies close to a region of junctional zone made up of complex basal lamina-enclosed astrocyte processes (a), collagen and small axons adjacent to the glia limitans of the parenchymal border zone overlying an astrocyte cell body (top left). Scale bars: (a) 1.0 p~m,(b) 0.5 p~m. Fig. 22. Very small putative axonal sprouts (arrows) within the proximal graft. Some of the sprouts display small focal contacts with glial processes. Scale bar = 0.25 ~m. Figs 23, 24. One column dominated by astrocyte processes (a) (Fig. 23) and another by Schwann cells (S) (Fig. 24). Both columns contain a few axonal sprouts (arrows), are enclosed in basal lamina and surrounded by fibroblast processes. Scale bar = 0.5 p~m.

772 astrocyte and S c h w a n n cell processes (Figs 14, 16). Both types of astrocyte c o l u m n usually contained some axons. Astrocyte processes were still p r e s e n t in the graft at two m o n t h s (Figs 29, 30, 32). Astrocyte cell bodies were not seen in the graft at any stage. The axons associated with astrocyte processes w e r e usually small and in non-intimate contact with the astrocyte plasma m e m b r a n e (Figs 14, 17b, 23, 30) but some were large a n d invaginated the astrocyte plasma m e m b r a n e or w e r e partially e n w r a p p e d by astrocyte processes (Figs 16, 29). Astrocyte processes within these columns also sometimes e n w r a p p e d other astrocyte or S c h w a n n cell processes.

Regenerating axons. A few small axonal sprouts were p r e s e n t at the p e r i p h e r y of the graft at five days, but at eight days there w e r e small and m e d i u m - s i z e d axons p r e s e n t in superficial contact with either glial cell processes of uncertain origin (Fig. 9) or p r e s u m p t i v e S c h w a n n cell processes (Figs 10, 11). A minority of such axons was more intimately related to S c h w a n n cell processes (Fig. 12). At longer survival times the n u m b e r of axons increased, they i n v a d e d d e e p e r

CAMPBELL, LIEBERMAN, ANDERSON and TURMAINE portions of the graft (by 14 days) and more of t h e m became intimately e n w r a p p e d by S c h w a n n cell or astrocyte processes within basal lamina-enclosed S c h w a n n cell columns (Figs 15, 25), p r e d o m i n a n t l y astrocyte columns (Figs 17b, 23,,30) or mixed columns of astrocyte and S c h w a n n cell processes (Figs 16, 29). The regenerating axons were often located in the central parts of columns of S c h w a n n cell processes (Fig. 14) or mixed columns (Fig. 16) rather than adjacent to the basal lamina. At five, eight and 14 days, most axons in the graft were small, r o u n d and sprout-like with few organelles. At one and two m o n t h s , in addition to the small axonal sprouts, larger axons were present with m o d e r a t e n u m b e r s of organelles, sometimes including clusters of irregularly shaped, p r e d o m i n a n t l y flat vesicles resembling the vesicles of presynaptic terminals at Gray type 2 synapses in the CNS (Peters et al., 1991). The small axons, at all ages t e n d e d to have a non-intimate contact with S c h w a n n cell or astrocyte processes. T h e y were a p p o s e d to the plasma m e m b r a n e of one or more irregular S c h w a n n cell processes at a few small contact points (Figs 9-11, 14, 17c, 24), rather than

Fig. 25. One month after grafting. A region of the proximal graft in which Schwann cells (S) and mixed Schwann cell-astrocyte (a) columns are much more tightly packed than in Fig. 17 although they are similarly compartmentalized by processes of fibroblasts (f). Most of these columns contain axons (some arrowed) which are medium-sized and many of which are more deeply impressed into the Schwann cell than are the small axons illustrated in Figs 17c, 22 and 24. Scale bar = 1.0 ~m. Figs 26-33. Two months after grafting. Fig. 26. Survey electron micrograph of the interface between the parenchymal border zone (PBZ) and the graft (G) including a vascular, but not clearly demarcated, junctional zone (JZ) close to a complex glia limitans consisting of multilayered undulating astrocyte processes (large arrow). Note that many axons in the graft are now myelinated. Scale bar = 5.0 ~m. Figs 27, 28. High power micrographs of the complex, multilayered glia limitans between the parenchymal border zone and the junctional zone. It is made up of irregular astrocyte laminae (a) between which are sandwiched small nonmyelinated axons (some arrowed), other glial processes, and pockets of extracellular space containing collagen and proteinaceous material. Scale bars = 0.5 ~m.

Figs 29, 30. Nonmyelinated axons of variable size in basal lamina-enclosed columns within the graft. The larger column in Fig. 29 includes an intermediate filament-rich astrocyte process (a) and a Schwann cell (S), and is apparently penetrated (at left) by one of the fibroblast processes which surround the column (f). In the smaller column, the axons are associated with one or two astrocyte processes (a) and a thin process of uncertain identity (at right of bundle). Scale bars: (Fig. 29) 1.0 ~m, (Fig. 30) 0.5 p~m. Figs 31-33. Mature axons myelinated by Schwann cells (S, Fig. 32) and surrounded by thin fibroblast processes (f, Figs 32, 33), astrocyte processes (a, Fig. 32), a few small nonmyelinated axons (arrows, Figs 32, 33) and collagen. Scale bars: (Fig. 31) 2.0 ~m, (Figs 32, 33) 0.5 ~m. Figs 34-44. Electron micrographs of transverse sections through the distal graft and adjacent parenchymal border zone between five days and two months after implantation. Fig. 34. In Fig. 34a two large bundles of small axonal sprouts are seen in the junctional zone of the distal graft five days after implantation. (See Fig. 2b for a photomicrograph of the interface between parenchymal border zone and graft in a corresponding semithin section). One bundle is transversely sectioned (small arrows) and lies amongst collagen between presumptive epineurial fibroblasts. The other bundle is longitudinally sectioned (between large arrows, and shown slightly rotated and at higher magnification in (b)) and appears to leave the parenchymal border zone (bottom left of figure) at a breach in the glia limitans and enter the junctional zone/epineurium. Scale bars: (a) 2.0 ~m, (b) 0.5 ~m. Fig. 35. Nonmyelinated axons (arrows) sandwiched between Schwann cell processes in the distal graft 14 days after implantation. Scale bar = 0.5 ~m. Fig. 36. A column of Schwann cell processes enwrapped in basal lamina and surrounded by thin fibroblast processes (f) in the graft 14 days after implantation. Within the column only one or two small irregularly shaped profiles (arrows) may be axons. Scale bar = 0.5 ~m.

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enwrapped by the processes. Small regenerating axons were often in contact with one another (Figs 9-11, 14, 17b, 30). The larger axons tended to be totally or partially enwrapped by Schwann cell processes (Figs 12, 15, 16, 25). Most of the larger axons were non-myelinated. A very few axons, myelinated by Schwann cells, appeared at one month, but at two months many were myelinated particularly in one animal (GC61) in which the graft was dominated by mature Schwann cell-myelinated axons of medium to large size (Figs 26, 31-33). In the other two month animal (GC84) only a few myelinated axons were present and the graft was dominated by small to medium-sized unmyelinated axons generally in intimate contact with Schwann cells. (Fig. 29). OBSERVATIONS BY ELECTRON MICROSCOPY: DISTAL GRAFT

The parenchymal border zone The parenchymal border zone was similar to that around the proximal graft at comparable survival times. It was covered by an incomplete glia limitans at 5-8 days (Fig. 34) which with longer survival times developed more layers and became more complete and complex, containing interdigitating layers of collagen, macrophages, Schwann cell processes and thin astrocyte sheets (Figs 40, 41). Beneath the glia limitans lay clusters of sprout-like profiles, often in contact with astrocytes, oligodendrocytes or microglia. The sprouts resembled those in the parenchymal border zone around the proximal graft in terms of their size and shape, their relationships to glia and other neural elements, and because there were larger numbers present at 5-8 days than at one and two months. There were more large nonmyelinated axons with an irregular shape than in the parenchymal border zone around the proximal graft, perhaps representing the normal contingent of unmyelinated cortical axons, which are more numerous than in the thalamus, especially in the

most superficial part of the cortex (layer I; plexiform layer). Degeneration debris contained within microglia and astrocytes, and a few reactive neurites were present, particularly at 5-8 days but were hardly apparent at two months. The remainder of the parenchymal border zone consisted of areas of normal neuropil and a few myelinated axons.

The junctional zone The junctional zone associated with the distal graft was very different from that adjacent to the proximal graft in that it resembled (and probably consisted principally of) peripheral nerve epineurium. Between the reforming/reformed glia limitans of the brain and the perineurium of the graft were layers of elongate fibroblasts and fibroblast-like cells with a basal lamina, amongst large collagen fibre bundles often coursing in different directions (Figs 37, 40, 41). Sandwiched between the fibroblast layers were elongate macrophages and at one and two months there were a few Schwann cells and nonmyelinated axons, some of which appeared to be undergoing degeneration (Fig. 40). At two months a few Schwann cell-myelinated axons were present within the junctional zone. At 5-8 days this epineurium-like tissue was more disorganised than at later survival times and close to the glia limitans was often intermixed with CNS components such as astrocytes, nonmyelinated axonal sprouts and oligodendrocyte myelinated axons (Fig. 34). Some of the axonal sprouts were organised into large bundles apparently traversing the interface between brain parenchyma and junctional zone (Fig. 34). The graft General appearance. The distal graft closely resembled the proximal graft; basal lamina-enwrapped Schwann cell columns, many containing macrophages and myelin debris, and some containing regenerating axons and empty basal lamina tubes were set in a

Figs 37--40. One month after grafting. Fig. 37. Interface between parenchymal border zone (PBZ) and graft (G) showing the nature of the connective tissue sheaths that enwrap the graft. (See Fig. 2d for a photomicrograph of the same interface in a semithin section). The vascular epineurium (EP) comprises widely spaced layers of fibroblasts with interspersed macrophages and Schwann cells. It separates the glia limitans (GL) from the tightly packed layers of the perineurium (P). Within the graft is a single myelinated axon (double arrow) among the Schwann cells and myelin debris. Scale bar = 5.0 ~m. Figs 38, 39. Small nonmyelinated axons (some arrowed), in the distal graft. The axons are loosely associated with Schwann cell processes in basal lamina-enwrapped columns. Two Schwann cell bodies are present in the column in Fig. 38, one of which is labelled (S). Scale bar = 0.5 ~m. Fig. 40. (a) Interface between the multilayered glia limitans of the parenchymal border zone, consisting of irregular astrocyte sheets and fingers (a) and the junctional zone/epineurium which is made up of fibroblasts in a heavily collagenous matrix. Between the fine fibroblast processes is a small, basal lamina-enwrapped Schwann cell column (open arrow), shown at higher magnification in Fig. 40b, which contains a few degenerate nonmyelinated axons (arrows in (b)). These axons have interrupted plasma membranes, and electron lucent cytoplasm containing small, irregularly-shaped vesicles. Scale bars: (a) 1.0 p~m,(b) 0.5 p~m.

CNS axon regeneration into PNS grafts collagenous matrix ,(Figs 38, 39, 44). The distal graft differed from the proximal graft in the absence, at any stage, of astrocyte processes and in the presence of a distinct perineurium (Figs 37, 41, 42). Also at 14 days and later survival times the distal graft contained less debris and fewer debris-laden macrophages than the proximal graft (compare Fig. 2c with 2d).

The perineurium. At all survival times the perineurium was made up of approximately ten layers of tightly packed, thin, basal lamina-enclosed cells (Figs 37, 41). Some nonmyelinated axons and Schwann cells were sandwiched between the layers of perineurial cells, particularly between the more superficial laminae. At 5-8 days soma of these nonmyelinated axons closely resembled axonal sprouts, and were sometimes present in large bundles between the innermost as well as more superficial cellular laminae.

Discussion

General remarks This study provides a detailed ultrastructural account of the regenerative growth of adult mammalian CNS axons into peripheral nerve grafts. We have shown that within days of implantation of sciatic nerve autografts into the thalamus of adult rats large numbers of axonal sprouts accumulate within the zone of damaged brain tissue surrounding the graft (parenchymal border zone) and subsequently in the most peripheral part of the proximal portion of the graft itself (junctional zone) which by eight days contains large numbers of such sprouts, some of which are already loosely associated with Schwann cell processes. By two weeks after grafting many sprouts have penetrated deep into the graft, most have established at least a superficial contact with a Schwann cell process, and some have developed a more intimate relationship with Schwann cell processes, giving rise

779 to Schwann cell axon bundles similar to those in regenerating peripheral nerves. Many axons have also established contact with astrocyte processes. The fastest growing axons reach the distal graft at 8-14 days and by four weeks numerous regenerating axons have reached the distal graft. Between one and two months after implantation many of the regenerating axons have acquired myelin sheaths. The thalamus was selected as the site of graft implantation for the following reasons: (i) pilot studies undertaken prior to the present studies, in which sciatic nerve autografts were placed in the cerebral cortex (with or without simultaneous lesion of the corpus callosum), indicated that the cerebral cortex was an unsuitable site for a study of this kind since in 11 experiments the number of neurons that could be retrogradely labelled from the distal tip of the graft 4-12 weeks after implantation ranged from two to a maximum of 12 in agreement with the studies of Benfey and Aguayo (1982); (ii) previous studies by Benfey and colleagues (1985) had indicated that sciatic grafts placed in the thalamus of adult rats are readily invaded by CNS axons; (iii) our laboratory is experienced in the ultrastructural analysis of thalamic neuropil (e.g. Lieberman & Webster, 1974; Spaeek & Lieberman, 1974; So et al., 1985; Campbell & Frost, 1988).

Identity of small nonmyelinated profiles. We believe that the small nonmyelinated elements are newly regenerated axonal sprouts. Our reasons are as follows. They have the ultrastructural characteristics of developing CNS axons (Henrikson & Vaughn, 1974; Peters et al., 1991); regrowing early postnatal rat retinal ganglion cell axons (Dyson et al., 1988; Taylor et al., 1989); axonal sprouts at the tip of the retinal stump of transected optic nerve in rat, identified as retinal in origin by anterograde labelling (Richardson et al., 1982); and regenerating adult rat retinal ganglion cell axons in peripheral nerve grafts (Hall & Berry, 1989).

Figs 41-44. Two months after grafting. Fig. 41. Survey electron micrograph demonstrating the constituents of the graft (G) and parenchymal border zone (PBZ) and, between them, the connective tissue sheaths of the graft. The graft still contains large clusters of macrophage-engulfed myelin debris among the myeIinated axons and Schwann cells and is enveloped in a perineurium (P) made up of closely spaced cell laminae separated by collagen. The graft is separated from brain tissue by a thick, vascular epineurium (EP) consisting predominantly of large bundles of collagen, and towards the brain, widely separated layers of fibroblasts with interspersed collagen, macrophages and mononuclear cells. The thick, multilayered glia limitans (GL) separates the peripheral nerve components from the thinly myelinated axons of the CNS parenchymal border zone. Scale bar = 10 b~m. Fig. 42. Branch of a peroneal nerve graft displaying many myelinated axons, Schwann cells and some myelin debris, separated from the main nerve (upper left) by a collagefious, vascular epineurium, and a thin perineurium (between arrows). (See Fig. 2b for a photomicrograph of the main peroneal nerve and its two branches five days after grafting). Scale bar = 10 b~m. Figs 43, 44. Mature axons myelinated by Schwann cells and nonmyelinated axons partially enwrapped by Schwann cell processes (small arrows) or only focally apposed to Schwann cell cytoplasm (Fig. 44, arrowheads). The basal lamina-enwrapped Schwann cell columns lie among collagen and fibroblast processes (f) typical of peripheral nerve endoneurium. Scale bars: (Fig. 43) 0.5 ~m; (Fig. 44) 1.0 ~m.

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At the earliest survival times the sprouts are most numerous in the parenchymal border zone near the graft. Within the proximal graft they are restricted to the region closest to the brain parenchyma. Only at longer survival times do they enter central regions of the graft. They are more numerous than small nonmyelinated axons in equivalent thalamic regions contralateral to the graft or in control animals. And whereas the small nonmyelinated axons of normal thalamic tissue tend to be isolated or occur in small groups within the neuropil and among bundles of myelinated axons, in the parenchymal border zone around the graft the small nonmyelinated axonal profiles are frequently present in large clusters of up to several score. Furthermore, in thalamic parenchymal border zone tissue these profiles tend to have a smooth, rounded and commonly circular outline, whereas small nonmyelinated axonal profiles in the contralateral thalamus or in the thalamus of unoperated animals tend to be irregular in outline and moulded to adjacent neuropil components, almost as though they are squashed in a confined space. Examples of such sparsely distributed and irregularly shaped small nonmyelinated axons within the normal adult rodent thalamus are illustrated in numerous publications, e.g. lateral geniculate nucleus (Lund & Cunningham, 1972; Lieberman & Webster, 1974; So et al., 1985), lateral posterior nucleus (Crain & Hall, 1980), ventrobasal nucleus (Spaeek & Lieberman, 1974; Campbell & Frost, 1988). The differences between the nonmyelinated axons of the normal and grafted thalamus may reflect the fact that the parenchymal border zone around the graft tends to have abnormally large areas of extracellular space. In the developing brain the extracellular spaces are also more extensive and nonmyelinated axons are usually rounded (Karlsson, 1967; Campbell et al., 1984). Nonmyelinated axons of the normal adult CNS are often highly irregular in outline (for example see pp. 107, 111, 121, 133 and 161 in Peters et al., 1991). Further evidence that these elements are axonal sprouts comes from our finding that some ultrastructurally similar profiles in the parenchymal border zone 3-14 days after graft implantation are immunoreactive for the growth associated protein, GAP-43 (Campbell et al., 1991). The fact that GAP-43 has been shown to be present at high levels in regenerating axons of both the PNS (Skene & Willard, 1981b; Verhaagen et al., 1989; Tetzlaff et al., 1989) and the CNS (Skene & Willard, 1981a; Campbell et al., 1991), in addition to its prominence in axons and growth cones of developing neurons (for reviews see Skene, 1984, 1989; Benowitz & Routtenberg, 1987), suggests that GAP-43 may be a useful marker for growing axons and can be used as a tool for the identification, as in the present study, of regenerating axonal sprouts. However, there is some recent evidence which suggests that GAP-43 may not

be exclusively neuronal in localization (Vitkovic et aI., 1988; Tetzlaff et al., 1989; Woolf et al., 1990; da Cunha & Vitkovic, 1990; Campbell et al., 1991; Curtis et al., 1992).

Origin of sprouts There is very strong evidence that the sprouts in the brain parenchyma and at least most of those in the proximal graft in the first few weeks after graft implantation are of CNS origin, and a strong presumption that they derive from axotomized CNS neurons located close to the graft tip (see below). This is an important point because graft implantation is likely to damage perivascular and meningeal nerves (Andres et al., 1987) from which regenerating autonomic and sensory axons may enter the graft. Such PNS axons may enter the graft from the exposed and open distal end, through the holes in the epineurium and perineurium made by the suture thread that ties the graft to the dura mater, or perhaps directly through the epineurium and perineurium at other sites. It is also possible that graft insertion will damage vascular nerves of the pia mater and choroid plexus of the transverse fissure which is often close to the proximal end of the graft, or that strands of meningeal tissue will be forced into the brain at the time of graft implantation and thereby provide a potential source of regenerating peripheral axons from nerves pushed into the brain with the meningeal tissue. The sprouts within the brain parenchyma are almost certainly of central origin. The evidence is as follows. Large numbers of sprouts appear within the parenchymal border zone soon after graft implantation. They are present at the earliest postgraft survival times that we have observed, that is at three days (Campbell et al., 1991) and large numbers are present five days after grafting (see Results). The sprouts are restricted to the parenchymal border zone around the graft both proximally and distally and are not seen in such numbers or in such clusters in the normal brain parenchyma. We have not quantified the sprouting response of the parenchymal border zone to graft implantation but on purely qualitative grounds we can say that the number of sprouts in the parenchymal border zone close to the proximal graft reaches its maximum at 5-14 days post-graft survival and then declines. The thickness of the parenchymal border zone is'd crude indicator of the number of sprouts present within it. This zone is 100-150 ixm thick at 5-8 days and contains large numbers of sprouts, at two months it is 40-50 ~xm thick and contains considerably fewer sprouts. Penetration of the parenchymal border zone by regenerating axons of peripheral neurons in such vast numbers and at such an early stage after grafting is highly improbable. Furthermore, regenerating peripheral nerve axons avoid CNS tissue unless the glial cell population of the CNS has been depleted

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CNS axon regeneration into PNS grafts (Reier et aI., 1983; Anderson & Turmaine, 1986; Anderson et al., 1989). Those axons which do enter CNS tissue display a varicose appearance and are surrounded by astrocyte processes (Stensaas et al., 1987; Anderson et aI., 1989) unlike the parenchymal border zone sprouts in the present study. Finally, recent immunohistochemical work in this laboratory has demonstrated (by light microscopy) large bundles of GAP-43 positive sprouts entering the proximal graft from the parenchymal border zone during the second week after graft implantation (Vaudano et al., 1992a) at precisely the same time as GAP-43 immunoreactive neuronal somata become detectable adjacent to the graft tip (Campbell et al., 1991, 1992; Vaudano et aI., 1992a). Indeed very recently we have been able to trace individual GAP-43 immunoreactive axons from immunoreactive cell bodies into the graft (Vaudano, unpublished). It is thus highly probable that at least some of the sprouts arise from the GAP-43 immunoreactive neuronal somata. As for the sprouts within the graft itself, it is less easy to be certain that all are of central origin but it is probable that most are, for the following reasons. Sprouts have been observed in every part of the graft/brain interface, that is below, within and on top of the glia limitans, in all areas of the junctional zone, within the graft itself and traversing between brain and graft tissue. (Although the directionality cannot be determined from such EM observations, the immunohistochemical observations referred to above suggest that they are passing from brain into graft). Thus we have observed sprouts in all the sites one would find them if they were growing from brain to graft. It is unlikely that the sprouts were growing in the opposite direction, that is from graft to brain, because sprouts were present in the proximal graft at 5-8 days and it is improbable that any peripheral axons penetrating from the distal graft would have reached the level of the proximal graft at this stage. Furthermore, at 5-8 days after graft implantation most sprouts were located within the parenchymal border zone and the junctional zone, at 14 days they predominated in the junctional zone and periphery of the graft and only later (1-2 months) did the sprouts appear in substantial numbers in the central core of the proximal graft. Fate of regenerated CNS axons The important question of the long term fate of the CNS axons that are induced to regenerate into and along peripheral nerve grafts is unresolved by these observations and we are currently examining lor~ger term survival animals to clarify this issue. Preliminary observations suggest that there may be regression and a sharp reduction in the number of axons in the grafts after two months. This would not be surprising. Although we know that CNS axons regenerating along PNS grafts may show normal electrical activity

(Keirstead et al., 1985; Munz et al., 1985; Gauthier & Rasminsky, 1988), that under certain conditions a few may be induced to re-enter the CNS environment and establish functional synaptic contacts with appropriate targets (Vidal-Sanz et al., 1987; Carter et al., 1989; Keirstead et al., 1989), and that these contacts may endure for many months (Vidal-Sanz et aI., 1991), in our study, the axons within the graft (the distal end of which was simply left below the scalp) have no natural targets, indeed no obvious targets of any kind, with which to form trophically sustaining relationships. In this context it is relevant to recall the remarkable degree of maturation and myelination that takes place in the grafts by two months. This suggests that the regenerating axons derive adequate trophic support from the graft initially but that additional factor(s) are required for the long term maintenance of the regenerated axons (see also Gauthier & Rasminsky, 1988). Relationship of glial cells to the regenerating axons The majority of regenerating axons within the graft at 14 days and thereafter display specific or at least highly preferential cellular associations. In the proximal graft these associations are primarily with Schwann cell processes, and to a lesser extent w i t h astrocyte processes. In the distal graft they are with Schwann cells alone. However, at earlier times after grafting (5-8 days) axonal sprouts within the parenchymal border zone and the junctional zone appear not to be related in a consistent manner with specific cell types. That is, they were not seen to be intimately and commonly in contact with one particular cell type in the parenchymal border zone or as they pass from brain tissue to the junctional zone and graft~ We therefore conclude that regenerating CNS axons may not rely on any particular cellular substrate to guide them in their growth from brain to graft. Because of the known affinity between axons and Schwann cells and the close associations that are established between the regenerating CNS axons and Schwann ceils in the junctional zone and proximal graft from eight days onwards, we looked carefully for evidence that Schwann cells might be involved in guiding axons from the brain into the Schwann cell columns of the graft. Schwann cells do migrate from the bands of von Biingner of the proximal graft and are found between the pre-existing basal lamina tubes in the proximal graft as well as in the junctional zone, but this does not occur during the early stages of axonal outgrowth from the brain into the junctional zone. Furthermore we found no evidence that Schwann cells penetrate far into the brain in significant numbers. The latter observation is closely in line with observations of others (e.g. Berry et al., 1988b; Hall & Berry, 1989; Kuhlengel et aI., 1990) and with experimental evidence indI~cating that Schwann cells penetrate CNS tissue only after X-irradiation or other severe treatments

782 which deplete CNS glia (e.g. Gilmore & Duncan, 1968; Gilmore, 1973; Blakemore, 1976; Blakemore et aI., 1986). On the other hand, we do have some LM data (immunohistochemical observations with antibody against the low affinity NFG receptor: Vaudano et al., unpublished; see Vaudano et al., 1992b; Campbell et aI., 1992), which suggest that Schwann cells may penetrate the brain after insertion of peripheral nerve grafts. Furthermore, Schwann cells which entered the brain parenchyma would be difficult to identify by EM unless they secreted a basal lamina or enwrapped axons. Current evidence suggests that tropic and/or trophic molecules, emanating from the peripheral nerve graft (most likely from the Schwann cells; see Paino & Bunge, 1991) attract and support the regenerating axons. Certainly both injured or intact peripheral nerves can produce neurotrophic factors, including NGF, NT-3, CNTF and FGF (Williams et aI., 1984; Heumann et al., 1987; Maisonpierre et al., 1990; Eckenstein et aI., 1991; Sendtner et al., 1992). Furthermore, receptor molecules for some of these factors have been found on CNS neurons that are capable of regenerating axons into a peripheral nerve graft (Hagg et al., 1990, 1991; Wood et al., 1990; Carmignoto et al., 1991). Specific neurotrophic factors for thalamic neurons are u n k n o w n although their survival is promoted by diffusible factors arising from cerebral cortical neurons (Cunningham et al., 1987; Sharp & Gonzalez, 1986; Hisanaga & Sharp, 1990). Additional evidence for the support and attraction function of peripheral nervous tissue comes from recent experiments in which Schwann cells or small pieces of sciatic nerve placed into the vitreous body of the adult rodent eye induce axotomized retinal ganglion cells to sprout axon-like processes and increase the survival of retinal ganglion cells (Cho & So, 1989, 1992). This work suggests that some diffusible trophic material reaches the retinal ganglion cells from the intravitreal transplant. Interactions between regenerating axons and Schwann cells There appear to be two distinct types of association between axons regenerating into sciatic nerve grafts and Schwann cell processes. One is an intimate contact in which Schwann cell cytoplasm partially or totally enwraps the axon and large areas of axonal and Schwann cell plasma membranes are in close apposition. This type of contact is common at longer survival times and is often associated with larger axons. The second kind of relationship is one in which the axons, which are generally smaller and more irregularly shaped than those involved in the intimate type of relationship with Schwann cells, are not enwrapped by the Schwann cell processes but establish small areas of focal apposition with them. This

CAMPBELL, LIEBERMAN, ANDERSON and TURMAINE type of relationship is common at short survival times but can still be seen two months after grafting. The Schwann cell processes involved in these nonintimate relationships with axons resemble those seen in long term denervated peripheral nerves (Weinberg & Spencer, 1978). The lack of an intimate, enwrapping relationship between the Schwann cells and axons is reminiscent of the relationship between Schwann cells and axons during the initial stages of both development (Webster & Favilla, 1984) and regeneration (Anderson et al., 1983) of PNS axons. We do not know w h y some axons should be intimately enwrapped by Schwann cells and others have only a few contact points with Schwann cell membranes. It is unlikely that the non-intimate type of contact is associated with only recently regenerated axons because this type of contact is seen at two months after grafting in both proximal and distal graft. With regard to such axons, however, we do not know if they arrived at their final proximal and distal positions within a few days of grafting and have remained there for approximately two months with little change to their morphology, or if they have taken two months to arrive at their position, or finally if they are newly regenerated and have just appeared either at proximal or distal ends of the graft. This latter possibility seems unlikely, for the fastest axons regenerating from thalamic neurons seem to take only 8-14 days to reach the distal graft, a distance of approximately 7mm. This is a similar growth rate to that of the fastest regenerating retinal ganglion cell axons of the adult hamster which grow at a rate of 2.5 mm per day, after an initial delay of 4.5 days (Cho & So, 1987). It is clear that many axons regenerate at a much slower rate than this, since large numbers of axons are found in the distal graft only after one month, but to reach the distal graft after only two months would imply remarkably slow regeneration. Axons appear to instruct their associated Schwann cells to enwrap them (Aguayo et al., 1976; Weinberg & Spencer, 1976; Bunge, 1986; Jessen et al., 1987). It therefore seems likely that some regenerating CNS axons are capable of instructing Schwann cells to enwrap them while others cannot. Those axons with intimate Schwann cell contact are generally larger than axons with only a superficial Schwann cell relationship. This might indicate that an intimate relationship with a Schwann cell is required for axonal maturation. In this context it would be interesting to examine, in this experimental situation, expression of cell adhesion molecules involved in PNS axon-Schwann cell interactions during development and regeneration. Extension of astrocyte processes into the graft The presence of astrocyte processes in the peripheral region of the proximal graft, where they commonly appeared in basal-lamina-enclosed columns, which often also contained axons and Schwann cells, raises

CNS axon regeneration into PNS grafts an interesting question. Do astrocytes migrate from the parenchymal border zone into the graft, or are the astrocyte processes in the graft long extensions from astrocytes located at or just below the brain-graft interface? The migration of astrocytes from host CNS into graft tissue has been reported by Zhou and colleagues (1986) (from adult rat septum into transplanted superior cervical ganglion) and hinted at by Kuhlengel and colleagues (1990) (from neonatal rat spinal cord into implants of dorsal root ganglion cells and Schwann cells). However, in most reports of peripheral nerve implants into CNS tissue the predominant phenomenon illustrated is extension of astrocyte processes into the graft, not migration of astrocyte cell bodies (e.g. Weinberg & Raine, 1980; Chi et al., 1980; Chi & Dahl, 1983; Berry et al., 1988a; Hall & Berry, 1989). Our own observations offer no support for the bodily migration of astrocytes into the graft but suggest instead that the astrocyte processes in the graft are long finger-like extensions from the glia limitans, as originally described by Weinberg and Raine (1980). Thus astrocyte processes were concentrated in the peripheral portion of the proximal graft, and absent from the core of the proximal graft and from all parts of the distal graft. Furthermore, astrocyte cell bodies were never encountered within grafts. The absence of astrocytes in the distal graft further suggests that the perineurial sheath of the implanted peripheral nerve is an effective barrier to the penetration of the graft by astrocyte processes. We do not know h o w far along the graft the astrocyte processes extend, only that they do not extend as far as 6-7 mm. Presumably, in the columns containing axons and a mixture of astrocyte and Schwann cell processes, the astrocyte processes terminate (at distances of the order of scores or a few hundred micrometers from the tip of the graft), and are replaced by Schwann cell processes more distally.

783 parenchyma immediately around the graft, which, surprisingly, have not been recognized previously in similar studies. We have also described how these sprouts leave the brain to enter the junctional zone and the graft, and the associations they establish with Schwann cells as they regenerate into and along the graft. However, many significant questions remain unanswered. We have presented arguments that the regenerating axons in the graft, especially in the proximal graft after short post-grafting intervals, are predominantly of CNS origin, but we do not know the extent to which axons of peripheral origin are present in the proximal or distal graft at different intervals after grafting. And a satisfactory explanation is needed of the gross numerical disparity between the numbers of regenerating axons identified in the grafts and the much smaller numbers of retrogradely labelled neuronal cell bodies detected in LM-HRP studies (Morrow et al., 1993). Another area of ignorance relates to the extent to which the events we have described depend upon the presence of living cells in the graft. It will be important to examine the early responses to implantation of killed grafts and the placement of lesions without grafts. There are also unresolved issues concerning interactions between the regenerating CNS axons and central and peripheral glia. In particular, the strength of our conclusion that glial cells are not directly involved in the growth of sprouts from the brain into the junctional zone and graft needs to be further tested, for example by EM analysis of serial thin sections. And the immaturity of axon-Schwann cell relationships in the graft at 1-2 months raises questions about how prolonged the sprouting response is, and about abnormalities in the interactions between regenerating CNS axons a n d Schwann cells by comparison with those between regenerating PNS axons and Schwann cells. Work aimed at answering some of these questions is currently in progress.

Concluding remarks This study has shed some light on the early regenerative events that follow implantation of grafts of living peripheral nerve into the CNS. In particular, we have identified the early axonal sprouts within the brain

Acknowledgements We wish to thank Action Research for the Crippled Child and the Wellcome Trust for financial support.

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