Ultrastructural Studies of Dorsal Root Axons RegeneratingThrough Adult Frog Optic and Sciatic NervesROSA E. BLANCO,* JULIO ROSADO, JUAN PADILLA, AND CLARISSA DEL CUETOInstitute of Neurobiology and Departments of Anatomy and Neurosurgery, School of Medicine, University of Puerto Rico,San Juan, Puerto Rico

KEY WORDS Schwann cells; astrocytes; amphibia; sensory axons

ABSTRACT Optic nerves of adult fish and amphibia can successfully regenerate, in partbecause their glial cells, unlike those of mammals, provide an environment permissive to regrowth.We altered the environment of regenerating dorsal root axons in the frog, Rana pipiens, by graftingsegments of optic nerve to test the permissiveness of CNS glial cells to other sensory neurons. Wecompared these preparations to grafts of segments of sciatic nerve. After allowing various times forsurvival, light and electron microscopy were used to evaluate the grafts. An agglomeration ofastrocytes, tightly joined by desmosomes, initially formed in the center of the optic nerve grafts.Around this grew regenerating dorsal root axons, accompanied by Schwann cells. At early stages,some axons formed dilated terminal structures, which were not seen in peripheral nerve grafts. Theappearance of blood vessels within the graft and the dispersion of cells allowed larger numbers ofaxons to grow through the graft. By eight weeks, 48% of dorsal root sensory axons had grownthrough optic nerve grafts, compared to 84% for sciatic nerve. These results suggest that astrocytesfrom optic nerve are not inhibitory to, and provide a suitable substrate for, regrowing sensoryneurons. Microsc. Res. Tech. 46:310–318, 1999. r 1999 Wiley-Liss, Inc.

INTRODUCTIONNeuronal processes in the mammalian peripheral

nervous system (PNS) regenerate after injury, whilethose of the central nervous system (CNS) do not. Thisis not due to intrinsic differences between CNS andPNS neurons, because axons within the adult mamma-lian CNS are capable of regenerative growth alongsegments of implanted peripheral nerves (So andAguayo, 1985). It is now generally believed that theglial microenvironment influences the success of regen-eration. Thus, Schwann cells of the mammalian periph-eral nervous system (PNS) are permissive to axonaloutgrowth (reviewed inAguayo, 1985; Baehr and Bunge,1989; Xu et al., 1997), while their myelin-formingcounterparts in the CNS, oligodendrocytes, are not(Schwab and Caroni, 1988).

Mammalian astrocytes are the predominant cell typein glial scars after injury (Reier et al., 1983) and it isbelieved that these so-called ‘‘reactive astrocytes’’ ob-struct axonal growth (Liuzzi and Lasek, 1987; Ridet etal., 1997). However, there is also evidence that imma-ture mammalian astrocytes can support axonal regen-eration (Rudge and Silver, 1990; Wunderlich et al.,1994). Cultured astrocytes that were transplanted toPNS were capable of sustaining axonal regrowth forseveral weeks (Hall et al., 1991). To date, no differencesin cell surface molecules have been found that couldaccount for this difference in growth-promoting proper-ties between neonatal astrocytes and reactive adultastrocytes (Hirsch and Bahr, 1999).

In contrast to mammals, optic nerve axons of fish andamphibia can regenerate after injury. This appears tobe due, at least in part, to a glial environment that ismore conducive to axonal regrowth. For example, axo-

nal regeneration of goldfish retinal ganglion cells isassociated with glial migration to the injured site(Blaugrund et al., 1993). Oligodendrocytes from fishand amphibian optic nerve support growth of regenerat-ing axons (Bastmeyer et al., 1991, 1994; Lang et al.,1995). Goldfish optic nerve myelin lacks the growthinhibitory proteins that are present in mammalianmyelin (Wanner et al., 1995). It has also been shownthat goldfish optic nerve contains two low molecularweight factors that promote outgrowth from retinalganglion cells in culture (Schwalb, 1996). Goldfisholigodendrocytes express an L1-like adhesion moleculethat promotes the growth of both fish and rat retinalganglion cells (Ankerhold et al., 1998).

We have previously examined the newly formedregion of outgrowth from the cut optic nerve of the frog,R. pipiens (Blanco and Orkand, 1996). These regenerat-ing axons are always surrounded by astrocytes, suggest-ing that these cells play a guiding or nurturing role thatmay contribute to the successful regeneration of theoptic nerve in lower vertebrates. We were, therefore,interested in studying whether frog optic nerve astro-cytes could also provide a permissive environment forregenerating axons of the peripheral nervous system.Frog lumbar dorsal root axons are capable of regenera-tion after cutting and reanastomosing (Liuzzi andLasek, 1985). We have taken advantage of this model totest whether glial cells from the optic nerve are permis-sive to regeneration of other types of sensory axons.

Contract grant sponsor: NIH; Contract grant numbers: NIH-MBRS SO6GM008224, NIH-RCMI G12 RR-03051.

*Correspondence to: Dr. Rosa Blanco, Institute of Neurobiology, 201 Boulevarddel Valle, San Juan, PR 00901. E-mail: rblanco@neurobio.upr.clu.edu

Received 9 December 1998; accepted in revised form 6 May 1999

MICROSCOPY RESEARCH AND TECHNIQUE 46:310–318 (1999)

r 1999 WILEY-LISS, INC.

Some of these results have been presented in abstractform (Blanco et al., 1995).

MATERIALS AND METHODSAdult frogs (R. pipiens) were used in the present

study. All the animals were treated according to theNIH Guide for the care and use of laboratory animals.Under tricaine anesthesia, the right eyeball was ap-proached via an incision in the palate; the extraocularmuscles were teased aside and a section of the intraor-bital part of the optic nerve was taken to be trans-planted. Dorsal skin and muscle were cut and thelumbar vertebrae exposed. A laminectomy was per-formed with the aid of a dental drill and ronguers. Asegment of a lumbar dorsal root (VIII), aproximately 2mm in length, was removed and a segment of opticnerve of approximately the same size inserted andattached with Ethicon 10.0 sutures. In another set ofanimals, a segment of sciatic nerve was used as acontrol transplant. All grafts were autografts. Afterdorsal root transection the animal maintained theipsilateral leg extended. The operated frogs were main-tained in separate tanks at 16°C, feeding on crickets.No efforts were made to systematically assess reflexes,but it was observed that the majority of animals couldhop normally between 6 and 8 weeks after the trans-plant.

Three animals per group were sacrificed at 2, 4, and 8weeks. Fixative containing 1% glutaraldehyde and 1%paraformaldehyde in 0.1M cacodylate buffer was per-fused through the heart, or poured onto the exposednerve, which was left in fix overnight. The proximalstumps were then carefully dissected, post-fixed withosmium, dehydrated, and embedded in Epon-Aralditefor semi-thin and thin sectioning. The sections werestudied by light (LM) and electron microscopy (EM).

RESULTSTwo to four weeks after the operation, light micros-

copy of sections of the transplant near the end of theproximal dorsal root (Fig. 1) showed that outgrowth ofmyelinated fibers had occurred (Fig. 2A,B). Many of theregenerating dorsal root axons were dilated, forminglarge bulbous structures (Fig. 2A,B). Lymphocytes and

macrophage-like cells were clustered amongst the axonsin the graft (Fig. 2B).

Electron micrography of the bulbous structures atthe ends of the regenerating axons showed that theycontained accumulations of neurofilaments and smoothendoplasmic reticulum, and were surrounded bySchwann cells (Fig. 2C,D). Bundles of small, unmyelin-ated axons were also present in the graft, some of whichcontained clusters of electron-lucent and electron-dense vesicles (Fig. 2E,F). These axon bundles werealso closely associated with Schwann cells (Fig. 2E,F).

At two weeks, the central portion of the graft ap-peared to have a compact core of cells surrounded byconnective tissue and blood vessels (Figs. 1, 3A). Elec-tron microscopic examination of this core region showedthat most of the cells were tightly apposed astrocytes,identified by their abundant intermediate filaments(Fig. 3B). The astrocytes were joined by numerousdesmosomes (Fig. 3B). Some cells, tentatively identifiedas macrophages, contained secondary lysosomes, a signof phagocytic activity (Fig. 3B). Myelin debris was alsopresent in this dense central region of the optic nervegraft, but no axonal processes were observed.

The central agglomeration of astrocytes was sur-rounded by a looser, peripheral region containing abun-dant collagen fibrils (Fig. 3D) and some cells showingevidence of mitotic activity (Fig. 3C). Some myelinatedaxons and Schwann cells were also present in thisperipheral region (Fig. 3C), and in some of the two-week preparations a few unmyelinated axons were alsofound at the border of the central astrocyte mass.

By four weeks, the central mass of astrocytes ap-peared to be more dispersed and less well defined.Myelinated and unmyelinated axons were observedcrossing the periphery of the graft (Fig. 3E).

At eight weeks, numerous bundles of regenerateddorsal root axons traversed the graft, embedded in acollagenous matrix (Fig. 4A,B). No bulbous axonalswellings were observed. Larger axons were myelin-ated and bundles of smaller axons were wrapped inglial cell processes (Fig. 4B), although in some casesthis wrapping was incomplete and the axons weresurrounded by extracellular matrix (Fig. 4C). In someaxons clusters of small, electron-lucent vesicles were

Fig. 1. Camera lucida drawing of a typical optic nerve graft 2weeks after the operation. Solid vertical arrows indicate the interfacesof the graft with the proximal and distal stumps of the dorsal root. Thedashed vertical line indicates the position from which axon countswere made. Only the largest, usually myelinated, axons have been

drawn; bundles of smaller axons are present in the lightly shadedareas. Some axons (ax) are shown growing around the central astro-cyte mass (As). Axonal swellings in the proximal stump and graft areindicated (sw) and blood vessels are shown (bv). Scale bar 5 0.5 mm.

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observed (Fig. 4C). After eight weeks, in some prepara-tions it was still possible to identify cells that exhibitedthe morphological characteristics of astrocytes, such asabundant intermediate filaments and desmosome junc-tions. These formed a meshwork around myelinatedand unmyelinated axons, and contained numerouscaveolae-like structures (Fig. 4D).

Counts of axons that had traversed the graft (Fig. 1)were made from ultrathin cross-sections. In three experi-mental animals, 3,508 6 222 axons, 851 6 40 (24%) ofwhich were myelinated, had crossed into the distal endof the graft. For comparison, counts of three normaldorsal roots, taken at the same level, showed 7,362 6695 axons, 2,842 6 250 (39%) of which were myelin-ated. Thus, approximately 48% of axons were able tocross the optic nerve grafts, and of these only 24% weremyelinated, compared to 39% in normal roots.

Regeneration of Peripheral Nerve RootsThrough Sciatic Nerve Transplants

Two to four weeks after the operation, electron micros-copy of sections through the central region of theperipheral nerve graft showed numerous myelinatedand unmyelinated axons, Schwann cells, and collag-enous extracellular matrix (Fig. 5A,B). No bulbousaxonal swellings were observed, but some myelin de-bris was present (Fig. 5B).

At eight weeks, the graft contained many moreaxons. Large axons were myelinated; some bundles ofsmall axons were wrapped by Schwann cell processesbut many axons persisted that were not completelyenclosed (Fig. 5C,D). Counts of transverse sectionsof the distal part of three grafts showed 6,218 6 865axons, 1,491 6 61 (24%) of which were myelinated.This indicates that approximately 84% of axons wereable to cross peripheral nerve grafts, and of theseonly 24% were myelinated compared to 39% in normalroots.

DISCUSSIONSensory Axons Regenerate Better Through

Grafts of Peripheral Nerve Than Optic NerveIn the present study, we have compared the effective-

ness of optic nerve grafts and sciatic nerve grafts inallowing regeneration of sensory axons. After eightweeks regeneration counts of axons revealed that ap-proximately 48% of the sensory axons had passedthrough the optic nerve grafts, whereas peripheralnerve grafts allowed almost twice as many (84%) toregenerate. Interestingly, the same proportion of these

axons (24%) was myelinated in both cases. What arethe possible reasons for these differences?

Astrocytic Aggregate Forms a TemporaryPhysical Barrier in Optic Nerve Grafts

In the early stages after the transplant, the majorityof cells that remained were astrocytes, which wereaggregated into a dense mass with numerous desmo-somes between them. This increase in the numbers ofdesmosomes has been previously reported during theformation of an all-glial nerve, 16 weeks after retinalablation (Blanco et al., 1993). While aggregation ofastrocytes can in part be due to a reaction to damage asa consequence of suturing (Hall and Kent, 1987), wesaw no such aggregation at the junction of graft andnerve stump. Instead, the astrocytes were clustered inthe central region of the graft, suggesting simply that,in the absence of axons to keep them apart, adhesionbetween them causes them to aggregate. During opticnerve regeneration astrocytes form fascicles around theregenerating bundles, maintaining contact with eachother (Blanco and Orkand, 1996).

This central mass of adhering astrocytes was noteasily penetrated by sensory axons. Axons were able toenter the proximal region of the grafts but there manyformed bulbous swellings containing neurofilamentsand other organelles, which were without myelin butwere surrounded by Schwann cells. It has been sug-gested that these morphological features are typical ofaxons that have been physically obstructed (Liuzzi andLasek, 1992). Axons were, however, able to pass aroundthe edges of this central astrocyte mass. In contrast,sciatic nerve grafts at two weeks contained no axonalswellings and allowed the free penetration of sensoryaxons. By four weeks, as the central astrocyte massbecame looser and smaller, myelinated and unmyelin-ated axons were able to pass through the center of thegraft and the axonal swellings also disappeared. Thus,while our results show that frog astrocytes appear toform a temporary physical obstruction to axonal re-growth, there is no evidence that they have any long-term inhibitory effects, as has been reported in mam-mals (Anderson et al., 1989; Hall and Kent, 1987;Liuzzi and Lasek, 1987), where sensory axons may beactively inhibited by contact with the astrocytes, pre-venting their penetration of the graft. In fact, there isevidence that Xenopus myelin and oligodendrocytesfrom optic nerve are not inhibitory to neurite extensionin vitro (Lang et al., 1995). Similarly, astrocytic glialscars from adult Xenopus transplanted to optic nervesdo not present a barrier to axonal regrowth, since these

Fig. 2. A: Longitudinal 1µm resin section of the proximal end of anoptic nerve graft into the dorsal root, 2 weeks after the operation.Several axonal swellings are visible (asterisks). Monocytes (arrow-head) and macrophages (arrow) are present. B: Same region of opticnerve graft, showing axonal swellings (asterisk) and numerous plasmacells (arrowheads). C: Electron micrograph of an axonal swelling inthe proximal end of the optic nerve graft. The swelling is surroundedby Schwann cells (s). Other axons are myelinated. D: Higher magnifi-cation electron micrograph of axonal swelling, showing the accumula-tion of neurofilaments (arrows). E: A small bundle of unmyelinatedaxons, some of which contain dense-core vesicles, is closely apposed toa Schwann cell (s). F: An axon terminal with an accumulation of clearand electron-dense vesicles (asterisk) is surrounded by myelinatedaxons of normal appearance. Scale bars 5 (A,B) 20µm; (C) 4 µm; (D) 1µm; (E) 4 µm; (F) 1 µm.

Fig. 3. (overleaf) A: Light micrograph of the accumulation ofastrocytes in the center of the optic nerve graft. A blood vessel (arrow)has invaded the extracellular matrix (asterisk) which surrounds theastrocyte mass. B: Electron micrograph of the astrocyte mass, showingtheir characteristic intermediate filaments (asterisk) and desmosomes(arrowheads). A macrophage-like cell (m) is present. C: Outside theborder of the astrocyte mass, a cell is undergoing mitosis (arrow) andmyelinated and unmyelinated axons are associated with Schwanncells. D: Unmyelinated axons (a) are present in the extracellularmatrix outside the border of the astrocyte mass (arrow). E: Electronmicrograph at 4 weeks after the operation, myelinated and unmyelin-ated axons were observed crossing the periphery of the graft. Scalebars 5 (A) 20 ım; (B–E) 2 µm.

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Fig. 3. (Caption on page 313)

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Fig. 4. A: Light micrograph of the central region of the optic nervegraft, 8 weeks after the operation. Large bundles of axons (shortarrow) course through the extracellular matrix and are surrounded byblood vessels (arrowheads). B: Electron micrograph of a bundle ofregenerating axons in the optic nerve graft at 8 weeks. Myelinatedaxons and numerous unmyelinated axons are associated with Schwann

cells and abundant extracellular matrix. C: Some of the small,unmyelinated axons contain vesicles (asterisk) and are not completelyenclosed by glial cell processes (arrows). D: Some cells resembleastrocytes, with abundant intermediate filaments and desmosomejunctions (arrow). They formed a meshwork around myelinated andunmyelinated axons. Scale bars 5 (A) 20 µm; (B,C) 2 µm; (D) 1 µm.

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are the appropriate axonal companions (Reier, 1979;Reier et al., 1983).

Role of Glial Cells in Promoting Sensory NerveRegeneration

Transplanted Schwann cells provide favorable condi-tions for regeneration of rat CNS axons, as well as thosein peripheral nerves (Xu et al., 1997). MammalianSchwann cells secrete neurotrophic factors, express celladhesion molecules, and generate numerous extracel-luar matrix molecules that influence nerve fiber growth(Guenard et al., 1993). In addition to the lack of anyphysical barrier, it is possible that frog sensory axonsregenerate almost twice as successfully through sciaticnerve, vs. optic nerve grafts, because the Schwann cellssupply them with growth-promoting substances thatare not provided by astrocytes or oligodendrocytes.

We were unable to identify unequivocally any oligo-dendrocytes in the frog optic nerve grafts; however, wecannot rule out the possibility that they are present.The myelin-forming glial cells that were observed in thecentral region of the optic nerve grafts were tentativelyidentified as Schwann cells, rather than oligodendro-cytes, because they formed a basal lamina and their cellbodies were directly apposed to the axons they en-sheathed (Peters et al., 1976). This would imply thatthey had migrated out from the ends of the graft. Aclarification of the possible role of oligodendrocytes inpromoting regeneration through the grafts will requirethe use of immunocytochemical markers, which arecurrently under development.

Whether the astrocytes in the frog optic nerve ac-tively promote the regeneration of sensory axons isopen to question. Immature astrocytes from rat opticnerve are capable of stimulating the regeneration of cutoptic nerve fibers of adult rats (Sievers et al., 1995).These activated astrocytes produce a multitude offactors, which may either inhibit or stimulate neuronalsurvival and process outgrowth (Muller et al., 1995).Frog astrocytes are facilitatory to the regeneration ofoptic nerve axons, which actually are observed in closeapposition to astrocytic processes (Blanco and Orkand,1996). They also express basic fibroblast growth factor(bFGF), which has a neurotrophic action when appliedto the optic nerve (Blanco et al., 1997). Results from anumber of studies indicate that astrocytes expressreceptors for growth factors and cytokines, suggestingthat the functional status of astrocytes may be subjectto regulation by these factors (Yong, 1996). Recently, ithas been shown that neuritic outgrowth from dorsalroot ganglion explants in three-dimensional astrocytecultures is greatly increased by treatment with interleu-kin-1a and bFGF (Fok-Seang et al., 1998). In ourexperiments, one striking feature of the response to

optic nerve grafts is the appearance of blood vesselswithin the graft. Angiogenesis is known to occur duringwound healing and may be important for tissue repairand growth (Liuzzi and Miller, 1990), and in this casemay promote the regeneration of the axons.

In conclusion, frog optic nerve astrocytes do notinhibit the regeneration of dorsal root sensory axons.Instead, the aggregation of astrocytes temporarily formsa partial physical barrier. As this barrier disappears,axons, accompanied by Schwann cells, readily regrowthrough optic nerve grafts, as they do through periph-eral nerve grafts.

ACKNOWLEDGMENTSThe authors thank Dr. Jonathan Blagburn for critical

reading of the manuscript. R.E.B. is supported byNIH-MBRS SO6 GM008224 and NIH-RCMI G12 RR-03051, and is grateful for the use of Histology Corefacilities supported by NIH-NINDS PO1 NS07464.

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