Differential effects of nerve, muscle, and fat tissue on regenerating nerve fibers in vivo

Axonal regeneration through silicone tubes was studied using distal nerve stumps, denervated, preatrophied muscle tissue, as well as fat tissue as a target. During the first stage of regeneration, i.e., within 2-3 weeks after surgery, a thin, filamentous structure consisting of fibrin and connective tissue was seen bridging the gap in all systems. Thereafter, this cord obvi- ously served as a guideline for the outgrowth of increasing numbers of axons into distal nerve stumps as well as into muscle tissue, but not into fat tissue. These findings confirm that preatrophied muscle tissue has a similar “neurotrophic” effect on regenerating nerve fibers as distal nerve stumps. The ineffectivity of fat tissue in promoting nerve fiber regeneration could be attributed either to the absence of “neurotrophic factors” or even to an inhibitory effect. Key words: nerve regeneration . muscle tissue fat tissue neurotropism nerve growth factors

MUSCLE & NERVE 12:723-734 1989

DIFFERENTIAL EFFECTS OF NERVE, MUSCLE, AND FAT TISSUE ON REGENERATING NERVE FIBERS IN VIVO

JOACHIM WEIS, MD, and J. MICHAEL SCHRODER, MD

Cylindrical silicone chambers bridging the gap between proximal and distal nerve stumps are a well-established experimental model (“tubulation experiment”) for the study of peripheral nerve fiber regeneration. Recent studies support the ear- lier observations of Forssmann12 and Caja14v5 that “neurotrophic factors” supplied by the distal stump of a transected nerve promote the outgrowth of regenerating nerve fibers from the proximal ~ t u r n p . ~ ~ ~ ” ~ ~ ~ Nerve fibers regenerating through silicone tubes showed conduction and cal- iber properties similar to nerve fibers that had grown through transected nerves reunified by epineurial suture.”” Also, the regenerating fibers showed formation of “normal” nodes of Kanvier.22 On the other hand, if the distal end of the chamber was ligated or left open, or if the distal nerve end was replaced by skin or tendon, no formation of a new nerve trunk bridging the gap

From the lnstitut fur Neuropathologie, Klinikum der RWTH Aachen, Fed- eral Republic of Germany.

Presented in part at the Joint Meeting of the Belgian, Dutch and German Societies of Neuropathology, October 9- 12, 1985, Aachen

Address reprint requests to Prof. Dr. J. M. Schrdder, lnstitut fur Neuro- pathoiogie, Klinikum der RWTH. Pauwelsstrasse, D-5100 Aachen. Fed- eral Republic of Germany.

Accepted for publication September 12, 1988

0148-639)(/1209/0723 $04.00112 0 1989 John Wiley & Sons, Inc.

was observed.44 Similar studies using the tubulation experiment for testing muscle or fat tissue as a target for regenerating nerve fibers have thus far not been performed.

In vitro experiments revealed that survival of spinal cord neurons is prolonged if they are sup- plemented with media conditioned by Schwann cells as well as skeletal or heart muscle cells.25 Sur- vival of cholinergic neurons is also su ported by extracts of denervated skeletal muscle.” Both media conditioned by muscle and extracts of skeletal muscle“ stimulated neurite outgrowth in vitro; moreover, media conditioned by muscle cells had a positive influence on neurofilament protein levels in spinal neurorial cultures.8 Hend- erson et al.” noted that the neurite-promoting activity of skeletal muscle was increased by denervation. On the other hand, extracts of muscle biopsies from patients with spinal muscular atrophies were inhibiting neurite outgrowth from spinal neuron^.'^ l’hese in vitro results are in accordance with the view that not only nervous tissue (e.g., peripheral nerve stumps or Schwann cells) but also skeletal muscle may promote regeneration of peripheral nerve fibers or produce “neurotrophic factors,” respectively. It is also well known from numerous studies that denervated muscle fibers induce terminal or collateral sprouting of nerve fibers in situ (see below). To test the influence of nerve, muscle, and fat tissue on peripheral

Regenerating Nerve Fibers MUSCLE & NERVE September 1989 723

nerve fiber regeneration comparatively under more standardized conditions in vivo, we used these tissues as distal inserts into nerve regeneration chambers.

MATERIALS AND METHODS

Fifty-four adult female Wistar rats weighing 200- 270 g were used for these studies: 16 for the tubulation of proximal to distal nerve stumps (N-N group = control group); 21 for the tubulation of proximal nerve stumps to distal, preatrophied muscle tissue (N-M group); and 17 for the tubulation of proximal nerve stumps to fat tissue (N-F group). The rats were held according to the law of protec- tion of animals in the Federal Kepublic of Germany (Tierschutzgesetz, 1986). The experiments were li- censed by the local authority (Regierungsprasident Koln; experiment No. 26.203.2-AC11).

The animals were deeply anesthetized by in- traperitoneal injection of chloralhydrate (35 mg/ ml) following ether anesthesia. The left sciatic nerve was mobilized by a dorsal incision at midthigh level; a small segment was resected, leav- ing a 6 mrn gap. Presterilized silicone tubes (hlohr KG, Aachen, FRG) with an inner diameter of 1.1 mm and an outer diameter of 1.8 mm were used to bridge the gap (Fig. 1). In all experimental animals, the tube was closed at the distal end with a Kocher's forceps, compressed by a dissecting forceps at midpoint level, and adjoined to the proximal nerve end. By opening the dissecting forceps, the nerve stump was drawn in rather atraumati- cally. The epineurium of the nerve end was sutured to the tube wall by 9.0 Prolene (Ethicon). In the N-N group (controls), the distal nerve stump was placed into the distal end of the tube and sutured as described above. In the N-M group, a portion of the distal half of the preatrophied bi- ceps femoris muscle was used in the same way following denervation and transection 10 days be- fore. In the N-F group, abdominal fat tissue was mobilized by Z-shaped incisions; a small segment, which was connected to the main portion of the fat tissue, was aspirated and sutured into the tube as described above. An empty space of approximately 6 mm within the silicone tube was left between the proximal and all distal inserts.

At 2, 4, 6, and 8 weeks after surgery, anesthetized rats of each group were sacrificed by in- traaortal perfusion with 3.9% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4.36 The silicone tube together with its contents and the proximally and distally located tissues were removed, post- fixed in a solution of' 2% phosphate-buffered os-

mium tetroxide, cut into pieces approximately 2 mm in length, and enibedded in epoxy resin.

Semithin sections from the proximal, interme- diate, and distal parts of' each tube were stained with paraphenylenediamine and photographed at comparable magnification. The area of the trans- verse-sectioned structures at the midpoint level of the tube was morphometrically evaluated by an in- teractive image analysis system (IBAS I, Zeiss, Oberkochen, FRG). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 400T electron microscope.

RESULTS

At 2 weeks post operationem ( p . ~ . ) , no significant differences between the three groups (N-N, N-M, and N-F) could be obscrved. A thin cord-like structure was seen bridging the gap in all tubes (Fig. 2a-c). The cord showed an hourglass shape with central tapering and in most chambers con- sisted of some layers of connective tissue elements and a few capillaries. In some chambers, only fibrin with sparse cellular elements connectrd the proximal and distal tissues. No nerve fibers were present at midpoint level. At the distal end, thickening of the cord was observed, resulting from cells migrating or growing in from the distal tissue. In N-N systems, Schwann cells were seen in this thickening originating from the distal nerve stump which included numerous bands of Bueng- ner.

At 4 weeks P.o., a nerve trunk consisting of myelinated and unmyelinated axons had regenerated across the gap in all N-N systems. At midpoint level, the trunk was ensheathed by a thin, imma- ture perineurium. In the distal nerve stumps, regenerating axons were numerous. They were accumulated in regions with a relatively high density of blood vessels (Fig. Zd, e). Few regenerating axons only had thus far reached the muscle tissue in 3 out of 4 N-M systems, although the gap briding cords at midpoint level in all instances contained many rnyelinated nerve fibers. In one distal insert (Fig. 3a, b) regenerating nerve fibers had grown in between preatrophied muscle fibers. Some nerve fibers were seen in a small, probably preexisting nerve fascicle. Others lay separate from muscle fibers and were usually grouped into minifascicles. The distal fat tissue was not invaded or penetrated by regenerating nerve fibers, although in two out of five chambers a thick gap bridging cord had been established containing numerous nerve fibers at midpoint level of the tube. Some nerve fi-

724 Regenerating Nerve Fibers MUSCLE & NERVE September 1989

REGENERATION CHAMBER

Nerve - Nerve

Proximal Distal

Nerve - Muscle

- _ - -

Proxi ma1 Distal

Nerve - Fat tissue

Proximal Distal FIGURE 1. Schematic view of the tubulation system used with the proximal nerve stump and the distal tissues: distal nerve stump, preatrophied muscle, or fat tissue.

bers, however, were found in the highly vascularized connective tissue that had reactively proliferated surrounding the distal fat cells. In between fat tissue, no axons were observed (Fig. 3c).

At 6 weeks P.o., in both the N-N and in most of the N-M systems, newly formed thick nerve fascicles with numerous nerve fibers were apparent at the midpoint level of the tube. Also, in the distal nerve stump as well as in the muscle tissue within

the distal end of these chambers, numerous regenerated nerve fibers were seen. Longitudinal sections revealed considerable enlargement of the distal cord close to the muscle tissue. Regenerating nerve fibers were distributed in small nerve fascicles between muscle fibers. In only one out of eight N-M chambers, the gap bridging cord was extraordinarily thin, and only a small number of axons had reached the muscle. In N-F systems,


FIGURE 2. (a) Proximal nerve stump in a N-M system 2 weeks after surgery. Numerous preexisting as well as regenerating nerve fibers and remnants of degraded myelin sheaths (arrows) are surrounded by an increased number of perineurial and epineurial cell layers. Bar = 100 pm. (b) A thin structured cord consisting of some layers of connective tissue and a few capillaries is apparent at the center. Bar = 50 prn. (c) Near the distal end, the central cord increases in thickness. Bar = 40 prn. (d) N-N system at 4 weeks after surgery. The gap is bridged by numerous cross-sectioned regenerating nerve fibers. The nerve trunk is ensheathed by a loose perineurium-like connective tissue. The axons appear to be accumulated at the sites of the larger blood vessels. Bar = 50 pm. (e) Distal nerve stump of the N-N system illustrated in d reinnervated by a large number of myelinated nerve fibers. The axons are arranged in small groups, presumably at sites of proliferated preexisting Schwann cells (bands of Buengner). Bar = 30 pm.

similar to those at 4 weeks P.o., numerous nerve fibers were apparent in the center of the chamber. Unlike the other N-F system, in one N-F chamber only, minimal nerve fiber regeneration across the whole gap was found. The fibers that had regen-

erated within this N-F system at midpoint level were ending, however, in the distal portion of the tube and had not invaded the fat tissue.

At 8 weeks P.o., a well-vascularized gap briding nerve trunk had been established in the N-N and


FIGURE 3. (a) N-M system 4 weeks p.0. The preatrophied muscle tissue at the distal end of the tube is highly vascularized. Bar = 100 pm. (b) At higher magnification of the area indicated in a, several minifascicles (arrows) consisting of hypomyelinated axons are visible between preatrophied muscle fibers. There are some regenerating nerve fibers in a small, probably preexisting nerve fascicle. A muscle spindle can be seen to the left of the nerve fascicle. Bar = 50 pm. (c) At the distal end of this N-F chamber, 4 weeks P.o., fat cells are surrounded by connective tissue. No regenerating nerve fibers are apparent between fat cells. Bar = 50 pm.


in the N-M chambers. In these systems, numerous regenerating nerve fibers had invaded the distal tissue (Fig. 4a, b). Electron microscopy (Fig. 5a, b) revealed small minifascicles of hypomyelinated

and unmyelinated axons surrounded by Schwann cells and by one or two layers of perineurial cells with incompletely developed basal laminae. Colla- gen fibers and fibrocytes were usually located be-

FIGURE 4. (a) Distal end of a N-M system, 8 weeks p.0. The nerve trunk invades the preatrophied muscle. Bar = 100 pm. (b) At a deeper level, the regenerating axons are usually grouped in minifascicles and distributed irregularly between the preatrophied muscle fibers. Bar = 50 pm.


FIGURE 5. Electron micrographs of the distal muscle tissue 8 weeks after surgery. (a) A minifascicle includes four hypomyelinated and several unmyelinated axons as well as several Schwann cells. The fascicle is demarcated by two to three layers of perineurial Cells (arrows) that are still incompletely covered by basal laminae (arrowheads). Thin collagen fibrils are distributed between these Cells and the surrounding muscle fibers. Bar = 2 pm. (b) Motor endplate with three nerve terminals (T) covered by several Schwann cell pro- cesses. Some irregular synaptic folds (arrows) in the muscle fiber and in the adjacent nerve fascicle are also present. Bar = 2 pm.

tween these fascicles and muscle fibers. Direct contacts between ingrowing axons and muscle fibers were only seen at the site of motor endplates (Fig. 5b). Usually, regenerating nerve fibers were well isolated from surrounding muscle fibers by inter- vening perineurial cells. In I%-F systems, 8 weeks P.o., regeneration had not significantly pro- gressed over what was seen already at 4 weeks p.0. A cord of variable thickness included at least some nerve fibers at proximal levels in all N-F systems, but no nerve fibers had invaded the distal fat tissue (Fig. 6).

Morphometry. The morphometric results are summarized in Fig. 7 and Table 1 . Two weeks after surgery, only small filamentous structures had bridged the gap between proximal nerve stumps and distal tissues in all systems. By the end of the fourth week, the cross-sectional area of these cords had increased in N-N and N-M as well as in N-F chambers. The latter varied considerably in thickness. Parallel to the increase of the number of nerve fibers, the cord progressively enlarged in the N-N and N-M systems during the sixth and seventh week after surgery. Thereafter, no further thickening of the gap bridging structure in the N-N and N-M chambers was observed. On the other hand, the cords in the N-F systems, at 6 and 8 weeks P.o., did not reach the values of cross-sectional area found in two chambers at 4 weeks after surgery. Usually, the gap-bridging structure in the N-F systems remained considerably thinner than in the N-N and N-M chambers.

Semiquantitative estimation of the number of nerve fibers at the end of the chambers (Table 1) revealed that no myelinated axon had reached the distal tissue at 2 weeks pm. At 4 weeks p.o., numerous regenerated axons had grown into all distal nerve stumps and into one out of four distally inserted muscles but not into fat tissue. 4 t 6 and 8 weeks after surgery, massive reinnervation was observed in nearly all N-N and most of the N-M chambers, whereas in the N-F systems no significant difference compared with the situation at 4 weeks after surgery could be found. In only 4 out of 10 N-F systems, a few axons had reached but not invaded the distal fat tissue (Table 1).

DISCUSSION

The present study revealed that not only distal nerve stumps but also preatrophied muscle tissue has a “trophic” influence on axonal regeneration within regeneration chambers in vivo. By contrast, in the N-F tubulation systems, there was only a

small number of nerve fibers bridging the gap and lack of ingrowing axons into fat tissue itself. Two stages of regeneration could be defined for both the N-N and N-M systems: during the first stage, within 2- 3 weeks after surgery, a gap-bridging cord consisting of connective tissue and capillaries was established by cell migration from either side of the regeneration chamber. During the second stage, i.e., 3-6 weeks after surgery, the cord was subsequently invaded by numerous axons. This second stage of regeneration, however, was absent or incomplete in all of the N-F chambers: only a few axons had reached the reactive connective tissue which surrounded the distally located fat tissue, and no axons were observed between fat cells.

The “neurotrophic” effect of preatrophied skeletal muscle observed in the present experiments resembles that of distal nerve stumps which has previously been studied rather extensive- ly.4,5, 12,27,32 Presencc of nervous tissue at the distal end of the tube was assumed to be essential for nerve regeneration inside chamber ~ y s t e m s . ~ ~ ~ ” ~ ~ ’ The present study leads to the conclusion that similar neurotrophic factors as in distal nerve stumps are also originating from preatrophied muscle.

Diffusible substances were proposed to medi- ate the neurotrophic effect in N-N regeneration

The results of the in vitro experiments mentioned in the Introduction revealed neurotrophic effects also of media conditioned by muscle cells and skeletal muscle extracts, respectively. In addition, studies on collateral sprouting of axons in denervated m u ~ c l e ~ * ~ ~ ’ ~ * supported the hypothesis that inactive muscle liberates neurotrophic factor( s).

chambers ,23,24,27,32

The Two Stages of Regeneration. As a result of the present study, two stages of regeneration could be distinguished. The first stage was observed in all systems, the second only in the N-N and N-M chambers. During the first stage, i.e., within the first 2-3 weeks, a gap bridging cord consisting of connective tissue was established in all three systems which apparently served as a guideline for regenerating axons. Even in some N-F chambers, a thick strand of connective tissue bridging the gap could be found (Fig. 7) , indicat- ing that this first stage of regeneration does not depend on the distal nerve stump or muscle tissue inserted into the distal end of the tube. The time when the first outgrowing axons (“pioneer fibers”42) reached the distal tissue (at approxi-


FIGURE 6. N-F system 8 weeks p.0. (a) Proximal nerve stump with numerous hypomyelinated, regenerating nerve fibers. Bar = 100 pm. (b) A thin, fibrotic cord bridges the gap at midpoint level. Bar = 10 pm. (c) Close to the distal end of the tube, the cord consists of connective tissue with some capillaries and several layers of sheath cells at the periphery. There are no regenerating nerve fibers. Bar = 10 pm. (d) The fat tissue at the distal end of the chamber contains no regenerating nerve fibers. Bar = 50 pm.

0. 3mm2

0. 2mm2

3.llIfll'

2 4 6 a Weeks a f t e r surgery

FIGURE 7. Diagram summarizing the planimetrically evaluated cross-sectional areas of all cords bridging the gap in the regeneration chamber at midpoint level. Single values are indicated by smaller symbols and mean values by larger symbols. There is an increase in cross-sectional areas up to the fourth week p.0. in all three systems. The gap-bridging structures in the N-N and .N-M systems, on the average, show continuous thickening up to the sixth week. In the N-F chambers, however, mean values at 6 and 8 weeks are much lower than in N-N and N-M systems.

mately three weeks p.0.) was regarded as the end of the first stage.

These observations are largely in accordance with the experiments of Williams et al.43 concern- ing formation of the cord in N-N systems. They found a thin coaxial fibrin matrix to be the first gap bridging structure. At 2 weeks p.o., Schwann cells, fibroblasts, and eridothelial cells migrated into the cord from both stumps. Even if the distal end of the chamber was left open, a granulation

tissue bridge along the gap was observed." No matrix formed, however, if a piece of skin was implanted distally. Components of' the cord in N-N systems at 14 days p.0. stained with antifibronectin and antilaminin,z3 Fibroncctin and laminin have been shown to promote or guide neurite outgrowth on various substrate^.^^^^^^^ In the experiments of Madison et a1.,28 a laminin-containing gel filled into N-N regeneration chambers were found to increase the rate of ingrowing axom.

During the second stage of regeneration, ingrowth of the majority of axons into distal nerve stumps or preatrophied niuscle and thickening of the nerve trunk within the gap was observed. The onset of the second stagc of regeneration in the N-M system, however, was somewhat retarded: 4 weeks after surgery, ingrowth of axons into preatrophied muscle was present only in 1 out of 4 N- M systems, whereas regcneration was already more advanced in all N-N systems studied. This can tentatively be attributed to a retraction of the preatrophied muscle tissue inside the chamber owing to some degree of scarification. Muscle tissue usually appeared to be traumatized more se- verely than nerve tissue during surgery. Increased gap length, however, is likely to delay ingrowth of axons into the distal tissue.24 On the other hand, ingrowth of axons into the distal nerve stump could also have been accelerated by Schwann cells migrating into the tube from the distal nerve stump arid thus forming a well-accepted guideline for outgrowing axons.

The second stage of regeneration was not observed in the N-F systems of the present series. In

14,36

~

Table 1. Myelinated nerve fibers at the distal chamber end (semiquantitative evaluation).

N-N N-M N-F

2 weeks p.0. 0

4 weeks p.0. +++

6 weeks p.0. +++ +++

0

+++ +++

+++ +++

8 weeks p.0. +++ +++ +++ +++ +++ +++ ++

0 0 0 0

+++ + 0 0

+++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ ++ ++

0 0

+ + 0 0 0 + o

+ + + o 0 0 0 0

Note: 0 = no regenerating axons in the disiai tissue; + = <20; + + = 20- 7 00; + + + = 1> 100; single symbols (+,O) or groups of symbols (+ +, + + +) refer to indwiduai iubulat/on sysiems; p.0. = post operationem.


this context, the hypothesis is proposed that effec- tive contact of initial regenerating nerve fibers (“pioneer fiber^"^') with distal nerve stunips or end organs, respectively, induces growth of more and more nerve fibers towards the target during the second stage. Growth could be promoted not only by mechanical or chemical contact along available channels but also by local or central elec- trophysiological signals or retrogradely transported, centrally operating substances or “neurotrophic hormones,”’ or “endogenous nerve growth

This hypothesis would be in accordance with the selection of neurons and nerve f i - bers that had reached their target tissue and the death of nonconnected neurons following equira- dial outgrowth of nerve fibers during development (Ref. 33, see also Ref. 35).

Recent studies revealed a neural cell adhesion molecule (N-CAM) present on surfaces of nerve fibers as well as on denervated and regenerating muscle fibers; it was, however, undetectable in nonsynaptic portions of normal, innervated muscle fibers.” Although we were not able to regularly observe direct contact of ingrowing nerve fibers with muscle fibers other than that seen at motor end plates (Fig. 5b), contacts to basal lamina components during early ingrowth of axons are likely to occur. Temporary interaction of growth cones with outer muscle specific surface components could play a role in attracting and guiding pioneer fibers. ‘l’his interaction would be somewhat differ- ent from the mechanism observed by Keynes et al.“ and <;lastly et al.,13 who found regenerating nerve fibers exclusively growing along the inner side of the basal lamina of muscle fibers treated with fornialiri fixation of freezing and thawing.

Nerve fiber outgrowth inside muscle fiber basement membranes was not a feature in the preatrophied muscle tissue used in our experiments: the regenerating nerve fibers were already grouped in minifascicles and, except at motor endplates, regularly surrounded by Schwann cells and perineurial cells with incomplete basal laminae at 6 and 8 weeks after surgery.

The relatively small number of gap-bridging

axons within the N-F systems, particularly the in- capability of regenerating nerve fibers to pene- trate the fat tissue at the distal end of the chamber, might be attributed to a lack of growth- promoting substances or a lack of fat cell surface- growth cone interactions, or even to the existence of antitrophic or “neurot.oxic” substances like the factors found by Manthorpe et ale3’ in glia cell-, heart-, and skeletal muscle-conditioned media and fluids from lesioned neural tissue.

In the only available report of nerve fiber growth into fat tissue up to the present time, Cajal“ described a “retarded” ingrowth of axons into a thin layer of fat tissue which was located between the two stumps of a transected nerve. The apparent growth-inhibiting effect of fat tissue in the present experimental system may be due to the length of the gap arid the lack of a distal nerve stump behind the fat tissue segment.

The inhibitory effect of fat tissue on regenerating nerve fibers was recently used as a means to experimentally prevent Iieuroma formatio~i.*~ Af- ter nerve dissection, the proximal nerve stump was implanted into fat tissue similar to the method of ‘l~enneff’i9 and Dellon et who implanted nerve stumps into musclc. There was a considerable limitation of axonal outgrowth, but formation of a solid neuroma, an end bulb rich in nerve fibers, could not be p re~en ted .~ ’

The Role of Blood Supply. Considering the relatively poor vascularization of fat tissue, the role of blood supply in influencing axonal outgrowth should be discussed. Although the granulation tissue encapping the fat cells at the distal end of the tube was well vascularized, it was invaded by a few axons only, if any. On the other hand, in distal nerve stumps and in reinnervated muscle, regenerated nerve fibers were found more frequently in the vicinity of blood vessels (Fig. 2b). This increase in vascular density could be a primary or second- ary phenomenon. ’l‘he blood supply is certainly a prerequisite for nerve fiber outgrowth in any case. But its irnport.ance should be established more clearly in future studies.

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