Axonal elongation through long acellular nerve segments depends on recruitment of phagocytic cells...

Brain Research 903 (2001) 185–197www.elsevier.com/ locate /bres

Research report

Axonal elongation through long acellular nerve segments depends onrecruitment of phagocytic cells from the near-nerve environment

Electrophysiological and morphological studies in the cata,b a,b a,b a˚Jesper Sørensen , Kare Fugleholm , Mihai Moldovan , Henning Schmalbruch ,

a ,*Christian KrarupaThe Institute of Medical Physiology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark

bDepartment of Clinical Neurophysiology, Rigshospitalet, Copenhagen, Denmark

Accepted 20 March 2001

Abstract

The distal nerve stump plays a central role in the regeneration of peripheral nerve but the relative importance of cellular and humoralfactors is not clear. We have studied this question by freezing the tibial nerve distal to a crush lesion in cat. The importance of constituentsfrom the near-nerve environment was assessed by modification of the contact between the tibial nerve and the environment. Silicone cuffs,containing electrodes for electrophysiological assessment of nerve regeneration, were placed around the tibial nerve distal to the crushsite. The interaction between long acellular frozen nerve segments (ANS) and the near-nerve environment was ascertained by breachingthe silicone cuff to allow access of cellular or humoral components. Tibial nerves were crushed and frozen for 40 mm and enclosed innerve cuffs with 0.45-mm holes or 2.0-mm holes to allow access of humoral factors or tissue ingrowth, respectively. In a second set ofexperiments, tibial nerves were crushed and either frozen for 20120 mm, leaving a 10 mm segment with viable cells in the center(stepping-stone segment) or frozen for 50 mm. These nerves were enclosed in cuffs with 2.0 mm holes corresponding to the viable nervesegment. The regeneration was monitored electrophysiologically by implanted electrodes and after 2 months the nerves were investigatedby light and electron microscopy. The results indicate that soluble substances in the near-nerve environment, such as nutrients, oxygen ortropic substances did not exert any independent beneficial effect on the outgrowing axons. However, phagocytic cells entering theacellular segment from the near-nerve environment were crucial for axonal outgrowth in long ANS. 2001 Published by ElsevierScience B.V.

Theme: Development and regeneration

Topic: Regeneration

Keywords: Nerve regeneration; Acellular nerve segment; Freeze lesion; Degeneration; Macrophage; Blood vessel

1. Introduction freeze lesion, leaving the connective tissue matrix intact[3,21,22]. Several experiments have demonstrated that

The processes taking place distal to an axotomy in a nerve fibers can regenerate through such acellular nerveperipheral nerve are Wallerian degeneration, axonal segments (ANS) [20]. However, without intrinsic cellulargrowth, reinnervation, and maturation. The first two pro- support, axonal elongation within long acellular nervecesses depend on actions of hematogenous phagocytic cells segments (ANS) may become increasingly dependent on[10] and Schwann cells [17,36]. factors in the near-nerve environment. Moreover there is

All cells in a nerve segment can be eliminated by a evidence that cells or soluble factors from the near-nerveenvironment improve axonal elongation in fluid-filledguidance channels (tubes) enclosed by a semipermeable

*Corresponding author. Department of Clinical Neurophysiology,membrane [1,2,14,24,25].Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark. Tel.: 145-

Our previous studies [20] suggested that a similar35-453-060; fax: 145-35-453-264.E-mail address: [email protected] (C. Krarup). influence from the near-nerve environment exists when

0006-8993/01/$ – see front matter 2001 Published by Elsevier Science B.V.PI I : S0006-8993( 01 )02441-6

186 J. Sørensen et al. / Brain Research 903 (2001) 185 –197

axons elongate through the structure of a distal ANS: for 2 min with silicone rubber-covered forceps [19]. Aaxonal elongation through a long ANS was possible, normal locomotion and behavioral pattern was resumedprovided that the nerve was left in its natural environment. 4–8 days after surgery. Sensory loss occurred on a smallHowever, when the ANS was shielded from the near-nerve area of the plantar surface; no harmful complicationsenvironment for a distance longer than 30 mm by an related to the injured nerves or to the implanted devicesimpermeable silicone tube, axonal elongation was marked- developed.ly impeded [20]. This suggested an interaction between the Freezing of the nerve comprised the crush site and afrozen endofascicular tissue and the near-nerve environ- segment of 40 mm or 50 mm distal to it (Fig. 1C). Duringment with implications for elongation through the long freezing the nerve was lifted from the underlying tissuesANS. The retarded regeneration in the long isolated ANS and a metal object, cooled in liquid nitrogen, was heldcould be related to impaired degenerative processes or to intermittently against the nerve for 5 min to achieve theabsence of necessary tropic factors or to both. effect of repeated freezing and thawing. This procedure has

In the present study, we have modulated the contact been shown to result in complete cell death while leavingbetween the near-nerve environment and 40 mm and 50 the extracellular matrix undamaged [22]. In the experi-mm ANS, in order (1) to confirm that an interaction ments designed to examine the influence of a limitedbetween the ANS and the near nerve environment has segment with viable tissue (Fig. 1C, stepping-stone, groupimplications for nerve regeneration, and (2) to determine III) [30], the nerve was frozen for 20 mm, followed by anthe relative importance of cells and soluble factors. unfrozen segment of 10 mm, and finally by a frozen

Axonal outgrowth was followed continuously for 2 segment of 20 mm in length distal to it. In the matchedmonths by the use of chronically implanted nerve cuff- experiment (Fig. 1C, group IV) the nerve was frozen forelectrodes, and at the end of the observation period the 50 mm distal to the crush site. Crush lesions werenerve was examined morphologically by light and electron performed at 0, 20 and 30 mm to delineate the stepping-microscopy. The results of the study indicate that recruited stone segment (Fig. 1C, group III) and in the matchedphagocytic cells are crucial for nerve regeneration through experiment (group IV) to avoid any bias introduced bylong ANS. multiple crush. The crush locations were marked with a

6-0 epineural suture for later identification.

2. Materials and methods2.3. Nerve cuffs and electrodes

2.1. Animals, surgery and postoperative careThe standard nerve cuffs were manufactured from

impermeable soft medical grade silastic tubes (MicroProbeThe experiments were approved by the national animal

Inc., Clarksburg, MD, USA), with stainless steel wireresearch committee. Thirteen young adult female cats with

electrodes (type 316 Cooner Sales Co., Chatsworth, CA,an average weight of 3.6 kg (Iffa–Credo, France) under-

USA) placed circumferentially on the inside of the cuffwent surgery in both hind limbs under aseptic conditions.

(2708 of a circle) [26]. The sciatic cuff had an internalAnesthesia was induced with intraperitoneal pentobarbital

diameter of 4 mm with six leads divided into two sets of(40 mg/kg bodyweight) and maintained intravenously.

three leads spaced 7.5 mm apart; the distance between theDuring surgery the animals rested on a water-heated

two leads in the center of the cuff was 3 mm (Fig. 1B).(378C) rubber pad to avoid hypothermia. The tibial nerves

The tibial nerve cuff had an internal diameter of 3 mmwere exposed over a distance of about 70 mm through an

with eight leads spaced 7.5 mm apart, and had a totalincision medial to the gastrocnemius muscle. After nerve

length of 54–55 mm. The tibial nerve cuff was modifiedcrush, freezing and implantation of the nerve cuffs and

before implantation to modulate interaction between theelectrodes (Fig. 1A), the wounds were closed in three

near-nerve environment and the ANS, (Fig. 1C). Afterlayers. Prophylactic Amoxicillin (150 mg, i.m.) was given

placement around the nerves, the cuffs were completelyperoperatively and 4 days postoperatively. The cats were

closed by external sutures and free sliding was ascertainedkept warm in an incubator until fully recovered from the

before the cuff was fixed by the surrounding tissue. Patchanesthesia. During further recovery, the cats were first kept

electrodes with two leads were implanted deep to plantarin a separate cage and then relocated to a common cage.

muscle together with a single electrode at the dorsum ofFood and water were available ad libitum and all cats

the foot to record evoked muscle action potentials (Fig.gained weight during the experiment.

1A,B). A ground electrode was implanted subcutaneouslyin the right hip region. All cables were insulated with

2.2. Nerve lesions teflon (except for the stimulation and recording sites),passed subcutaneously to the back, resurfaced through a

To ensure complete axotomy, a crush lesion of the tibial skin excision and soldered on a printed circuit board (Fig.nerve (Fig. 1A,B,C) was performed by clamping the nerve 1A). The circuit board, transcutaneously fixed to the spinal

J. Sørensen et al. / Brain Research 903 (2001) 185 –197 187

Fig. 1. (A) Overview of cat and chronically implanted electrodes. Cuffs were implanted around the tibial (T) and the sciatic (S) nerves andpatch-electrodes were attached to the plantar muscle (E) for EMG. Cables from the electrodes (C) were soldered into the connector-device (CC) mountedto the back. The tibial nerve was lesioned (LS) just proximal to the tibial nerve cuff-electrode. (B) Schematic drawing of the experimental set-up. Sixelectrodes were placed within the sciatic cuff and used in two tripolar recording configurations (R1 and R2); eight electrodes were placed within modifiedtibial cuff surrounding the nerve and used for stimulation of the outgrowing axons. The distances between all electrodes were 7.5 mm, except for 3 mmdistance between the adjacent electrodes at the two recording sites. The crush lesion that represents the proximal border of the ANS is indicated on thefigure. The distance between the crush lesion and the plantar muscle was about 140 mm. (C) In the four different experimental groups (I–IV), theinteraction between the near nerve environment and the acellular nerve segments (ANS) was modified. In group I, seven macroscopic holes (2.0 mm) alongthe silastic cuff allowed migration of cells from the near nerve environment into the ANS. In group II, the cuffs had 2.0 mm holes as in group I, but werecovered with a membrane with 0.45 mm pores. In group III / IV, four 2 mm holes in the center of the cuffs allowed both cells and solutes to support cellswithin the stepping stone segment (group III) or to enter at the mid-portion of the 50 mm ANS (group IV). C marks crush lesion (light gray), F marksfrozen section (gray). Unlesioned tibial nerve is black.

ligaments and muscles by 0-0 sutures enclosed in silicone 2.4. Experimental designtubes, allowed each lead to be connected to the electronicequipment for stimulation or recording as required. The animals were divided in four experimental groups


Table 1Experimental groups

Number of cats Right tibial nerve Left tibial nerve

6 I. Cuffs with 2 mm holes (n55) II. Cuffs with 0.45 mm pores (n55)5 III. Stepping stone experiment (n55) IV. Stepping stone control (n55)2 Cuff damage control (n52) Electrophysiological control (n52)

I–IV (Fig. 1C, Table 1). In group I, seven 2.0-mm holes potential (CNAP) was recorded at the two tripolar elec-were punched in the silastic cuff (Fig. 1C). In group II, trode configurations in the sciatic cuff (R1/R2, Fig. 1B),seven 2.0-mm holes were punched in the silastic cuff and amplified (15C02, Dantec, frequency range 200 Hz–6covered with a permeable teflon (polytetraflouoro-ethyl- kHz) and up to 500 traces were averaged (Nicolet 4094C).ene) membrane with 0.45-mm pores (Membrane TE-36, The stimulus was first applied to the proximal electrode,Schleicher & Schuell, Dassel, Germany) (Fig. 1C). Lasting and if an action potential could be elicited the stimulus sitepermeability of the membrane was confirmed at the end of was moved stepwise distally until a potential could nothe experiment by the diffusion of water-soluble Methyl- longer be recorded (500 responses averaged). The criteriaene Blue (5%) across the membrane. Cells from the to determine the presence of action potentials, have beennear-nerve environment were restricted from the ANS by defined previously [19,27]. The latency, amplitude, andthe membrane. In group III, four 2.0-mm holes were shape of the CNAP reflected the number, synchronizationpunched in the central part of the cuff corresponding to the and conduction velocities of the regenerated nerve fibersnon–frozen segment in the middle of the ANS (Fig. 1C). from which the action potentials were elicited [20].Viable tissue had access to the ANS via the 10-mm To detect the earliest reinnervation of the flexor musclesinterpositioned stepping stone segment. In group IV, the of the foot, a stimulus was applied to the sciatic and tibialcuff was designed as in group III, but with a 50-mm ANS nerves, and the compound muscle action potentialwithout stepping stone segment. (CMAP) was recorded from the plantar leads (Fig. 1A, B)

Experimental groups I1II (six cats /10 nerves) and III1 with a frequency range of 10 Hz–10 kHz.IV (five cats /10 nerves), respectively, were paired byperforming one experiment in the right tibial nerve and 2.6. Histologyanother in the left tibial nerve of the same cat. One nervein the original group I and one nerve in group II failed due Less than an hour after the last electrophysiologicalto technical problems during surgery or in the observation recordings, 2 months after the operation, the experimentalperiod. Hence two nerves from group I and group II was animals were sacrificed by intracardial injection of pen-matched, although they were not from the same cat. tobarbital during perfusion-fixation of the hind limbs

Two cats (four tibial nerves, Table 1) were used as through the abdominal aorta. Perfusion (200–300 ml /min)control for the electrophysiological procedures. was performed with an initial 7 min rinse (Ringer solution

with Heparin 5000 IE/ l and procaine 1 g/ l) under pressure22.5. Electrophysiology (0.25 Kp/cm ) followed by fixation for up to 10 min

(2.5% glutaraldehyde solution in cacodylate buffer andSerial observations started the day after implantation and distilled water 1:1). During perfusion both hind limbs were

were repeated every 3 to 4 days during the observation placed in the same position as used during the recordingperiod (59–63 days). At each recording session the cats sessions (908 hip and knee flexion and 908 ankle dorsiflex-were anesthetized with subcutaneous ketamine (10 mg/kg) ion). The nerves were exposed from the sciatic region toand xylazine (2 mg/kg). The temperature of the hind limbs the plantar muscles. All distances were measured and thewas kept constant by placing the cat on a heated rubber nerves were removed in toto. Additional fixation waspad (378C) and covering it with cotton. The temperatures performed in 2.5% glutaraldehyde for 24 h. Nerves wereof the plantar areas were measured at completion of the divided, postfixed in osmium tetroxide (1%, for 2 h), andelectrophysiological session; it ranged from 34.5 to 35.58C. embedded in epoxy resin (Embed 812, Electron Micro-The integrity of each lead in the cuffs was tested in situ scopic Science, Fort Washington, USA). Cross-sectionsbefore the recordings by measuring the impedance between 3–5 mm thick were cut with dry glass knives and stainedelectrodes and ground, the impedance usually ranged from with p-phenylenediamine. Thin sections for electron mi-0.5 to 1 KV. croscopy were cut from selected areas of the epoxy blocks

Excitable axons in the ANSs were identified by stimulat- and were stained with uranyl acetate and lead citrate. Theing consecutive leads in the tibial cuff. The electrical two control cats were sacrificed at day 28 by the samestimulus was a biphasic negative–positive pulse (duration procedure.0.1 ms) from a constant-current stimulator with an output Cross-sections of all experimental nerves were examinedup to 10 mA. The ascending compound nerve action by light microscopy at the crush site (level 0) and 15 mm,


30 mm, 45 mm and 60 mm (group III and IV) distal to the 2.8. Statisticslesion. For myelinated nerve fiber counts photomicrog-raphs were printed on a video printer (Sony Multiscan Results in numbers are generally given as mean6S.E.M.UP930) to final magnifications of 1103 (total cross-sec- Individual statistical tests are mentioned when used.tional area) and 7103 (sample) and assessed with the aidof a digitizer tablet (Wacom) connected to a computer.

2Three to four rectangular areas of 50,500 mm each were 3. Resultsrandomly sampled for myelinated nerve fiber counts. Intotal, the sampled area covered about 20% of the total 3.1. Electrophysiologycross-sectional area. The total number of fibers wascalculated from the mean density of myelinated fibers in Stimulation of the regenerating axons in the modifiedthe samples and the total endoneurial area. In nerves with tibial nerve cuffs elicited compound nerve action potentialsan obviously uneven fiber density between the fascicles, (CNAPs), which, when recorded in the sciatic cuff (Fig. 2each fascicle was evaluated individually. Blood vessels A, B), had amplitudes and shapes similar to those recordedwere counted on the total cross-sectional intrafascicular in earlier studies with impermeable standard cuffsarea of the nerve with the aid of a microscope equipped [19,20,26]. A compound muscle action potential (CMAP)with an ocular grid. Repeated counts of the same area of could be obtained from the plantar muscles when only athe same section varied by less than 2%. In three nerves in few immature nerve fibers had reached the muscle (Fig.group I, serial sections (50 sections with 25 mm interval) 2C). The CMAPs could be detected up to 10 days beforewere cut to trace newly formed blood vessels. Electron visible muscle contraction.microscopic evaluation (Phillips type CM 10, Holland) In all experimental situations, the first potentials couldwas performed in selected areas in nerves of group II as be elicited after an initial delay of 7–10 days (Fig. 3A, B).well as in the control nerves. After this, the elongation characteristics differed between

Due to the inability to mark macrophages by immuno- the experimental groups.histochemical stains in the cat, despite repeated attemptswith trans-specific and non-specific markers, identification 3.1.1. Cuffs with 2.0-mm holes (group I) compared toof phagocytic cells as either macrophages or Schwann cells cuffs with 0.45-mm holes (group II)could not be carried out in the light microscope. In group I (Fig. 3A) axons of all nerves elongated

through the 40-mm ANS, although with some variability(23–51 days after the lesion). The elongation rate was low

2.7. Control experiments in the ANS (0.8–1.7 mm/day), but increased to a rate ofabout 3 mm/day when the axons entered the non-frozen

The placement of holes in the nerve cuff changes the distal nerve segment to reach the plantar muscles in threedispersion of the stimulus current compared to our previ- of the nerves (48–57 days after the lesion). The observa-ous experiments with unbreached cuffs [19,20,26,27]. In tion time of 60 days did not allow an estimate of theorder to examine whether this would change the relation increase in elongation rate distal to the ANS in the last twobetween the front of regenerating sprouts and the site at nerves due to the missing time points for reinnervation ofwhich this front could be localized electrophysiologically, the plantar muscles. The best curve fit to describe thetwo lesioned and two unlesioned nerves with holes in the elongation in group I was polynomial (pooled data).cuffs (Fig. 1C, group I) were studied by electrophysiologi- In group II no axons could be detected distal to the ANScal recording and histological examination performed at at any time during the observation period. The elongationcorresponding levels within a few hours. The electro- rate in the ANS was 0.2 mm/day (linear curve fit, Fig.physiologically detectable front of regeneration was about 3A). The large short latency polyphasic CNAPs elicited at15 mm ahead of the site where fibers could be detected by the front of regeneration and the sudden disappearance oflight microscopy. Electron microscopy in addition revealed the potentials from one cathode to the next (Fig. 2B)thinly myelinated axons and unmyelinated sprouts at more indicated a delay in elongation which allowed the matura-distal sites, closer to the electrophysiological front of tion of fibers to catch up with elongation (compare withregeneration. Due to the spread of the electrical stimulus Fig. 2A).and the small number of fibers at the electrophysiologicallydetermined front, it was not possible to identify sprouts 3.1.2. Stepping stone 10 mm in 20120-mm ANS (groupwith certainty at the most distal electrode. The spread of III) compared to 50-mm ANS (group IV)the stimulus was probably slightly longer in these studies In group III, axons traversed the entire ANS in four outthan in earlier experiments [19,20], due to the breach of of five nerves (day 28–62 after the lesion). The regenera-the silicone insulation in these cuffs. However, serial tion in this group was not homogenous (Fig. 3B). Twoobservations were compared using similar cuffs with nerves re-innervated the plantar muscles (day 56 and 58)punched holes in all studies. and had a markedly different elongation rate (1.5 mm/day)


through the ANS than the other three (0.6 mm/day). Theelongation rate distal to the ANS for these two nervesincreased only marginally (Fig. 3B).

In group IV, elongation was similar in all nerves, with anelongation rate through the ANS of 0.3 mm/day (pooleddata, linear curve fit). Axons from two nerves elongated allthe way through the 50-mm ANS, but no plantar muscleswere re-innervated.

3.1.3. All groupsThe elongation rate through the ANS was reduced

corresponding to the degree of restriction of contactbetween the nerve and environment and to the length ofthe ANS. The best regeneration was seen in group I, withdecreasing elongation rates in group III, IV and II. Whenthe elongation slopes were tested by ANOVA followed by

´Scheffe’s test, group I was significantly different fromgroup II (P,0.01) and group IV (P,0.01), whereas theother groups did not differ significantly when assessed bythis test, due to the heterogeneity of elongation in groupIII, and the slow elongation in group II and IV.

3.2. Histology

3.2.1. Number of myelinated fibersIn all nerves, the number of myelinated fibers at the

crush level after 60 days exceeded the normal number atthis level (79836228, n510 [20]) by 10–20% due tosprouting (Fig. 4A). The sprouting at the crush leveltended to be more vigorous in nerves with impededelongation. The difference was significant between group I(89226396, n55) and group II (10,4476365, n55, P,

0.05, paired t-test), but not between group III (98026450,n55) and IV (95556542, n55), where the numbers wereintermediate compared to group I and II. At levels distal tothe crush lesion, the numbers were gradually decreasing,reflecting the distance below the crush level, as well as thedegree of contact restriction between the nerve and theenvironment (Fig. 4A). The highest numbers were found ingroup I, followed by group III, IV, and II, i.e., the sameorder as observed by electrophysiology.

3.2.2. Number of blood vesselsFig. 2. Action potentials from regenerating nerve fibers at different levels

In all groups, 60 days after surgery, the number of bloodof the ANS (A, B) and from plantar muscle (C). S5stimulus, n5numbervessels in the central part of the ANSs was smaller than inof responses averaged. Numbers at the left margin refer to the cathode

(Fig. 2) at which the stimulus was applied within the tibial cuff (A and B) non-injured control nerves with unbreached nerve cuffs.or to number of days after surgery (C). (A) Potentials obtained from a By contrast, the number of blood vessels distal to the ANStibial nerve (group III) 25 days after the lesion. The regenerating nerve was higher than normal in the nerve segments (Fig. 4B).fibers have reached the distal end of the ANS. (B) Potentials obtained

The number of blood vessels in the central part of the ANSfrom a tibial nerve (group II) 35 days after the lesion. The axonal(15 mm inside the ANS) was higher in group I than inelongation had reached approximately half way through the ANS

(between cathode 8 and 9). (C) EMG recordings at the plantar muscles group II, III and IV.during reinnervation (group I). A stimulus (S) was applied to sciaticnerve (cathode 6) and the EMG obtained by bipolar recording at the 3.2.3. Qualitative observationsplantar muscles (Fig. 2). At day 48, the first recordable muscle action

The groups with 2 mm vs. 0.45 mm holes in the cuff (Ipotential indicated reinnervation of a few muscle fibers. During theand II) were especially interesting because of the differen-following days the action potential gradually increased in size to produce

a visible movement of the paw at day 58. tial restriction of cells and soluble compounds, respective-


Fig. 3. Scatter plots (pooled data, five nerves in each group) to show elongation distance (ordinate) versus time (abscissa) relationship between group I(closed symbols, broken line) and II (open symbols, full line)(A) and between group III (closed symbols, no curve fit) and IV (open symbols, broken line)(B). (A) The best fit for group I was polynomial indicating different elongation rates inside and outside the ANS. The best fit for group II was linear.Squares mark the time of muscle reinnervation. (B) The heterogeneous elongation in group III excluded a meaningful curve fit. The best fit for group IVwas linear. Dots represent the time of muscle reinnervation.


Fig. 4. Diagram showing the number of myelinated nerve fibers (A) and blood vessels (B) (pooled data, five nerves in each group, except at *, where n54)at different levels distal to crushing 60 days after surgery. Error bars indicate6S.E.M. The dashed lines represent the average number of myelinated fibercounts (A) in normal tibial nerves (79836228, n510 [18]) and the number of blood vessels / square mm (B) in non-injured tibial nerves (12967.6, n56[18]). Experimental groups are indicated on the diagram. (A) The number of fibers reflect the degree of contact restriction between the ANS and thenear-nerve environment. The diagram also shows the sprouting at the crush level (0 mm), where numbers are 10–20% higher than control. (B) The figureshows that no blood vessels were found in the mid-portion of 40-mm ANS surrounded by a silicone cuff with membranes with 0.45 mm pores (group II),whereas revascularization took place when the 40-mm ANS were surrounded by cuffs with 2.0 mm holes (group I). The density of blood vessels was lowin group III and IV at 15 mm, but at 30 mm, corresponding to the stepping stone segment, the density was much higher in group III than in group IV. Theaverage density of blood vessels tended to be higher in the ends of the ANS than in the non-injured control nerves.


ly, and the large quantitative difference in regeneration ANS no living cells could be identified and the nervecapability demonstrated between the two groups. segment (4–6-mm-wide) appeared completely mummified

In the ANSs in group I, Wallerian degeneration was (Fig. 5A). On electron micrographs preserved parts of thealmost complete and only few phagocytic cells containing discontinued axon could be identified within the remnantsmyelin remnants and cellular debris were seen. Myelinated of the lamellar myelin sheets. Apparently no cell-mediatedregenerated nerve fibers and blood vessels were abundant degeneration had occurred during the 2 months followingthroughout the 40-mm ANS. On the contrary, in group II, the nerve injury (Fig. 5B). A transition zone (2–4-mm-Wallerian degeneration was only complete near the proxi- wide) containing myelin debris and phagocytic cells,mal and distal ends of the ANS. In the central zone of the delineated the mummified zone from the area where

Fig. 5. Electron micrographs from cross-sections of the mummified zone inside the mid-portion of an 40-mm ANS (group II), 60 days after the lesion(location of cross-section is marked in Fig. 7(*b)). Scale bars55 mm (A) and 1 mm (B). (A) No viable cells could be detected in the mummified zone.Apparently no cell-mediated degeneration had occurred during the 2 months following the nerve injury, and the collapsed myelin had only been subject toslight decay. (B) Preserved parts of the discontinued axon could be identified inside the remnants of the lamellar myelin sheets.


Wallerian degeneration was completed. A few millimeters examination of the 40-mm ANS in the 0.45 mm poreproximal to the transition zone, several thick myelinated experiment (this study) showed complete degeneration ataxons were present in the endoneurial tubes. The axons did both ends of the ANS, but not in the central part, thatnot enter the endoneurial tubes containing myelin debris. appeared mummified (Fig. 7). When the contact betweenIn the periphery, where the myelin had been removed the ANS and the near-nerve environment was not restrictedfaster than in the center, several myelinated nerve fibers to solutes alone, but included migration of cells from thewere present (Fig. 6A). environment (2.0-mm experiments, single or multiple

The blood vessels entering the nerves in group I (Fig. holes), degeneration occurred throughout the ANS. These6B,C) from the near-nerve environment through the 2.0- results demonstrate that the interaction with cells in themm holes were followed in serial sections to verify the near-nerve environment is crucial for initiation of de-penetration of the former perineurium in the ANS. The generative processes in the central segment of long ANSs.vessels, present as early as 14 days after surgery, traversedthe connective tissue strands into the holes and further 4.2. Regeneration of nerve fibers through long ANSsthrough the remnants of the perineurium into the intrafas-cicular endoneurial tissue, where they were seen to form Several observations indicate that Wallerian degenera-anastomoses with larger vessels. tion has to precede axonal regeneration: When a proximal

nerve stump is sutured to another proximal nerve stump,no regeneration takes place [9]. In the C57BL/Ola mouse,

4. Discussion where degeneration of the distal nerve stump is delayed,regeneration is sporadic [29] and in the normal nerve,

The findings of the present paper demonstrate inter- pre-degeneration enhances regeneration into acellularaction between long ANSs and the near-nerve environment nerve grafts [15]. In the present study, axonal outgrowthduring regeneration [20]. The extent of such interaction, only occurred in the part of the ANS that was cleared fromwhen modulated by different degrees of isolation of the myelin (Figs. 5 and 6A). In addition, electron micrographsANS, determined the extent of degeneration and regenera- of the ANS in the 0.45 mm experiment indicated thattion. The best axonal regeneration occurred when cells axonal sprouts do not enter a zone of intact myelin debris,from the near-nerve environment were allowed to enter the although large myelinated fibers have regenerated close toANS, and the histological investigations indicated that it. This impeded axonal elongation through a non-degener-invasion of phagocytic cells and blood vessels from the ated nerve segment may be due to simple physicalnear-nerve environment is critical for degeneration and obstruction of the endoneurial tubes by the tissue rem-regeneration in the long ANS. nants, or to actively inhibiting molecules present in the

myelin debris [34].4.1. Degenerative processes in long ANSs In the experiment with 2-mm holes in the middle of the

tube and 50 mm (group IV), axonal elongation tended toIn the unfrozen distal nerve segment, Wallerian degene- occur at longer distances than in the 0.45 mm experiments,

ration takes place by a concerted action of resident cells, and when combined with a short nerve segment withSchwann cells, and hematogenous non-resident phagocytic viable resident cells (group III, stepping stone), evencells, especially macrophages [10]. When a nerve graft is longer. The best regeneration, i.e. about up to 80% of theisolated from the environment by a millipore membrane, number of regenerated fibers detected in unshielded 40-impermeable to cells, no degeneration happens [8]. When a mm ANS [20], was observed in the experiments withdistal nerve segment is frozen, as in the present study, all 2-mm holes along the entire length of the ANS (Fig. 4A).resident cells and blood vessels are eliminated [3,21,23], Hence, our findings indicated that the regenerating capa-and the degenerative processes therefore rely on the supply bility of the axons corresponded closely to the level ofof Schwann cells and macrophages from the ends of the interaction allowed between the ANS and the near-nerveANS, as well as of phagocytic cells from the near-nerve environment, and thereby also to the degree of degenera-environment. We have to assume that these phagocytic tion in the ANS.cells from the near-nerve environment are macrophages, aswe were unable to label the phagocytic cells. This is in 4.3. Macrophages from the near-nerve environmentaccordance with the literature, as no reports of hemato-geneously recruited Schwann cells exist. The distance of Mononuclear cells of the macrophage system have beenthe degenerative processes, that can occur from the ends of identified as the main effector cells in myelin removalthe ANS in the cat, is 15–20 mm, as shown by shielding of during Wallerian degeneration [10] and by preventingthe ANS from the near-nerve environment [20]. However, macrophage invasion into a segment of a peripheral nerve,when the ANS is left unshielded in its natural environ- removal of myelin can be significantly retardedment, degeneration takes place throughout the segment, [8,12,15,29]. Macrophages are recruited in significanteven when the ANS is up to 70 mm long [20]. Histological numbers during the first days following a peripheral nerve


Fig. 6. (A) Light microscopic cross-section of part of a fascicle of a 40-mm ANS (group II), 60 days after the lesion (location of cross-section is marked inFig. 7(*a)). Scale bar5250 mm. Myelin debris has been removed from the periphery of the fascicle and several myelinated nerve fibers were present whereWallerian degeneration has occurred. In the central part of the fascicle, Wallerian degeneration has not occurred. This zone contained myelin debris andphagocytic cells, but no blood vessels or nerve fibers. (B) In situ macrograph of blood vessels and strands of connective tissue entering the cuff through the2.0 mm holes (group I), as it appeared before the tissue was removed for histological examination. The instrument tip points at the ingrowing tissue at themost distal of the seven holes in the cuff (only three holes can be seen). Scale bar55 mm. (C) Light microscopic cross-section of newly formed bloodvessels entering the ANS through the 2-mm-thick pedicle (arrows). On adjacent serial sections, several confluent blood vessels (*) could be followed fromthe periphery and into the endoneurium of individual fascicles. Scale bar51 mm.


sections in this study suggests, that even though the axonalregeneration appears unaffected by the lack of establish-ment of an epineural blood supply [7], regeneration ofnerve fibers depends on restoration of an endofascicularblood supply.

4.5. Stepping stone experiments

In situations where outgrowing axons must cross anenvironment without viable cells, the regenerative potential

Fig. 7. This figure summarizes the interpretation of the morphological can be increased when sources of viable Schwann cellsfindings within the endoneurium of a fascicle of a 40-mm ANS, 60 days (stepping stones) are introduced at intervals along path ofafter lesion (group I and II). Myelinated nerve fibers (black) traverse the

regeneration [4,30]. In this study we found the same effect,extent of the ANS in group I, whereas they are delayed by a zone ofbut with inconsistency. This variability may have beenmummified tissue in group II. A transition zone with progressingintroduced by a mismatch between the holes in the cuffdegeneration and macrophages (white with black bars) surrounds the

mummified tissue. Level of light micrographs (Fig. 6A) and electron and the stepping-stone segment (Fig. 1C), causing anmicrographs (Fig. 5A, B) are marked on the figure (*a and *b). Numbers insufficient support of oxygen and nutrients from theindicate distance (mm) below crush.

near-nerve environment.

injury and they migrate towards the degenerating axons4.6. Possible clinical impactand phagocytose myelin [35]. The mechanism underlying

this process appear to be related to the axonal disinte-In this study we have found that the interaction betweengration caused by calcium-activated proteases [11] perhaps

a long ANS and the near-nerve environment is critical toinitiated by monocyte chemoattractant protein-1 (MCA-1)the success of regeneration, and increasing degrees of[13]. Our study showed that by freezing the nerve, axonalrestriction of this interaction impede axonal regenerationdisintegration was severely impeded (Fig. 6B), and it iscorrespondingly. We assume that it will be equally im-possible that the lack of a specific chemotactic activationportant to secure the interaction with the near-nervesignal [34] prevents major recruitment of macrophagesenvironment in other long acellular grafts, natural orfrom the viable nerve ends alone (0.45-mm experiment).artificial. However, a possible benefit from using artificialOn the contrary, by exposing a large surface of the ANS tografts will be that no degeneration is needed beforethe environment immediately after freezing (2.0-mm ex-regeneration can take place. With extensive nerve lesions,periment), several macrophages may enter simultaneouslyit is sometimes necessary to use one or more long nerveand initiate a common activation-cascade [34] leading tografts. The need for donor nerves can in this situation be arapid myelin clearance. Furthermore, the macrophagesproblem, and studies of allografts [16,38], or artificialproduce factors [10] that stimulate Schwann cells andgrafts [5,6] have been carried out. One of the solutionsthereby influence regeneration of nerve fibers [17].could be cryopreserved allografts [38]. Our results showthat such preservation should be carried out after degenera-4.4. Ingrowth of blood vesselstion of the donor nerve in situ, as also suggested by Ochi etal. [32], to prevent mummification and retarded degenera-The revascularization of the long ANS takes place fromtion at the time of transplantation into the recipient site.the viable ends of the segment [20], and moreover from the

The need for a hematogenous source of phagocytic cells,near-nerve environment (Fig. 6B,C), across the epi- andstrongly suggested by our study, further emphasizes theperineurium. Such radial revascularization has also beenimportance of a well-vascularized bed for the long nerveobserved in long non-frozen nerve grafts [37], and seemsgrafts, especially if they are not predegenerated.to occur only when longitudinal revascularization cannot

prevent major ischaemia within the central part of the graftor ANS. The stimulation of angiogenesis occurs viaexpression of factors from cells in the ischaemic tissue Acknowledgements[33]. This is supported by our finding, that a source ofviable cells (stepping stone) left in the middle of the long We are grateful to Mrs Lis Hansen and Mrs MarianneANS improves revascularization at this site (Fig. 4B). Bjœrg for expert technical assistance. The work was

The longitudinal blood supply in the non-injured nerve supported by the Danish Medical Research Council, theconsists of two separate, functionally independent vascular Danish Research Academy, the Novo Foundation, thesystems, the epineural system and the fascicular system, Lundbeck Foundation, the Alice Brenaa Foundation, thewith extensive anastomosis between them [28,31]. The Leo Nielsen Foundation and the Foundation for Researchradial trans-perineurial revascularization detected in serial in Neurology.


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