Proc. Nati. Acad. Sci. USAVol. 86, pp. 1093-1097, February 1989Neurobiology

Individual microglia move rapidly and directly to nerve lesions inthe leech central nervous system

(nerve iinjury/ceil migration/video microscopy)

ELLEN MCGLADE-MCCULLOH, ALICE M. MORRISSEY, FERNANDO NORONA*, AND KENNETH J. MULLERDepartment of Physiology and Biophysics, University of Miami School of Medicine, Miami, FL 33101

Communicated by Gunther S. Stent, November 18, 1988

ABSTRACT Small cells called microglia, which collect atnerve lesions, were tracked as they moved within the leechnerve cord to crushes made minutes or hours before. The aimof this study was to determine whether microglia respond as agroup and move en masse or instead move individually, atdifferent rates, and whether they move along axons directly tothe lesion or take another route, such as along the edges of thenerve cord. Cell nuclei in living nerve cords were stained withHoechst 33258 dye and observed under dim ultraviolet illumi-nation using fluorescence optics, a low-light video camera, andcomputer-assisted signal enhancement. Muscular movementsof the cord were selectively reduced by bathing in 23 mMMgCI2. Regions of nerve cord within 300 ,um of the crush wereobserved for 2-6 hr. Only a fraction of microglia, typically<50%, moved at any time, traveling toward the lesion atspeeds up to 7 ,Im/min. Cells were moving as soon as obser-vation began, within 15 min of crushing, and traveled directlytoward the lesion along axons or axon tracts. Movements androles of leech microglia are compared with their vertebratecounterparts, which are also active and respond to nerveinjury.

Nerve injury triggers a characteristic sequence of cellularreactions. The early events occur whether or not regenera-tion follows. Thus, severed axons reseal and retract from thesite of lesion, which is rapidly populated by microglia andperhaps other phagocytic cells. There are several possiblesources and roles for microglia in regeneration (see, e.g.,refs. 1-5). Microglia may arise from blood or from nervoustissue itself, and while microglia are phagocytic, they mayplay additional roles. The goal of the present study has beento determine whether all microglial cells in the leech traveltoward the lesion and by what route they reach it.The leech nervous system has been useful for examining

neurons and glia during synapse regeneration (6). Leechmicroglia, like leech neurons and large glia, resemble theirmammalian counterparts in their morphology, physiology,and histochemistry (7-9). Moreover, resting and reactivemicroglia in the rat brain are selectively stained on theirsurfaces by a lectin from Griffonia simplicifolia seeds (10)that also in the leech stains microglia but not large glia andneurons (S. Hockfield, R. McKay, and K.J.M., unpublisheddata). Silver carbonate staining of cell bodies and Feulgenstaining of nuclei reveal that leech microglia normally liescattered among axons as an apparently homogeneous pop-ulation of cells within the nerve cord and collect at the site oflesion within 24 hr of nerve injury (9). The accumulationpeaks within 24 hr and declines slowly, never returning topreinjury levels, even after axons successfully regenerate.

Experiments in which nerve cords have been isolated intissue culture medium and then crushed have shown that

most, if not all, of the microglia at the lesion migrate therefrom within the nervous system (9). Although microgliaoccupy <5% ofthe volume ofthe nervous system, the gangliaalone are estimated each to contain 10,000-15,000 microglia(8), compared to only 400 neurons and 8 large glia perganglion (11). The axon bundles, or connectives, that linkganglia contain thousands of microglia yet have only 2 largeglial cells that each ensheathe -3000 axons. Furthermore, inthe connectives nearly all microglia are capable of moving tothe lesion, although usually they do not. In contrast, there isno apparent shift of cells of the perineurial sheath, which aredistinctive for their flattened rather than spindle-shapednuclei.An important question is how microglia within the nerve

cord reach the lesion. Do all microglia migrate toward the siteofinjury, moving en masse, or do only certain cells within theconnectives move? Is microglial migration chiefly at theedges of the connective along its outer surface, or do cellsanywhere within the connective move?

METHODSPreparation and Lesions. Segments of nerve cord were

removed from the animal and pinned in Sylgard-coated dishescontaining leech saline (12). Cords were stained 20-25 minwith Hoechst 33258 dye [0.001% (wt/vol) in leech saline] topermit tracking of microglial cells in the living connective.Hoechst 33342 dye stained similarly but was not used in theexperiments reported here. To optimize Hoechst staining ofthe cells and reduce background fluorescence, the tissue wasincubated overnight at 150C in Leibowitz-15 (L-15; GIBCO)tissue culture medium supplemented with 2% fetal calfserum, 0.6% glucose, and gentamycin (10 mg/ml) (13). Inother studies (14), it has been shown that nerve cordscultured in L-15 will grow for weeks and can regeneratesevered axons, which form new appropriate synapses. Con-nective segments 2-3 mm long were cut from the stainedcords between ganglia. Just before observation, the connec-tive segments were crushed with a pair of forceps (Dumontno. 5) ground to a width of 300 ,m, which severed the axons(15, 16). Except as noted, an isosmotic solution ofMgCl2 wasadded to the L-15 medium to bring [Mg2+] to 23 mM, therebyblocking spontaneous movements of the muscular connec-tives.Video Microscopy. In most experiments, a single region of

paired connectives 220 Am long and 50-200 ,um from thecrush was viewed with low-intensity epifluorescence illumi-nation (Zeiss filter set 487702) through a x40 water immer-sion objective at room temperature (20°C-23°C) for 2-6 hrafter the crush was made. The plane of focus passed throughthe centers of the connectives. Because bright UV lightarrested movement of stained cells (data not shown), theexcitation beam from the 100 W dc mercury arc lamp was

*Present address: School of Medicine, Universidad Central delCaribe, Cayey, Puerto Rico.

1093

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1094 Neurobiology: McGlade-McCulloh et al. Po.Nt.Aa.Si S 6(99

attenuated by up to 3 log units and a low-light video camera(model 66/ISIT; Dage-MTI, Michigan City, IN) used to viewthe preparation. Under these conditions, stained cells movedin response to injury. At these low light levels, the fluorescentcells were not visible to the unaided eye. With each exper-imental preparation, a stained and crushed control nervecord, not illuminated, was processed to confirm that low lightlevels did not affect microglia accumulation at the crush. Toimprove the contrast and signal/noise ratio of the videoimage, signals were processed with an Image I/AT imageprocessor (Universal Imaging Corporation, Media, PA).Processed and direct images were recorded with videotaperecorders (Panasonic NV-8050, Panasonic NV-8350, or JVCHR-S7000U).

Histology. After recording, the tissue was immediatelyfixed in Carnoy's fixative and processed for Feulgen staining(9). Stained crushed cords in the same recording dish, but notilluminated, were also processed. In addition to the unillu-minated controls, other controls were run to test the effectsof the Hoescht dye and elevated [Mg"~] on the accumulationof microglia, at the crush. The muscular nerve cords length-ened upon fixation in Carnoy's fixative, shrinking their lateraldimensions. In experiments and controls, many fewer mi-croglia accumulated at cuts, such as those made to isolate thenerve cords for tissue culture, than at crushes.Other nerve cords were stained by the del Rio Hortega

silver carbonate technique (see, e.g., ref. 17), which stainsmicroglia cell bodies, with results similar to those of Morgeseet al. (9). Thus, nerve cords were pinned with glass pins toSylgard 184 (Dow-Corning), fixed 4-28 days in 4% parafor-maldehyde, and then postfixed overnight in formol/ammo-nium bromide. After washing, the tissue was impregnated 1-2 min in 10% aqueous silver carbonate, reduced 1 min in 0.4%paraformaldehyde, toned 30 sec in 0.2% gold chloride, fixed3 min in 5% sodium thiosulfate, dehydrated, and mountedwhole.

RESULTSStained Microglia Accumulate at Lesions in Magnesium.

One step in tracking microglia was first to test whether theHoechst dye used to stain nuclei of living cells and Mg2l usedto block contractions interfered with microglia, accumulationat lesions. Some nerve cords were stained with Hoechst dyeand connective segments were cut from the cords betweenganglia. These Hoechst-stained segments of the connectiveswere then crushed, placed into culture medium containing 23MM Mg2l for ':-18 hr to allow microglia to accumulate atlesions, and fixed for Feulgen staining (as in Fig. 1). Othernerve cords were prepared as for the tracking experiments(see Methods). These stained segments were placed in tissueculture medium for up to 24 hr to optimize Hoechst staining,then crushed and incubated in culture medium containing 23MM Mg2l for an additional 18 hr. Other nerve cords wereeither stained with Hoechst dye and incubated in culturemedium without added Mg2+ or were not stained but wereincubated in elevated Mg2 . Controls, not stained withHoechst dye, were crushed and maintained in culture me-dium without added Mg2+. At least two cords were preparedfor each treatment.

Microglia accumulation was similar in the five treatmentgroups described above. Fig. 1 shows that microglia accu-mulated at the crush, reaching similar levels whether theyhad been stained with dye and treated with elevated Mg2+ ornot, and that these accumulations resembled those seen byMorgese et al. (9). The high density of cells at the crush andvariability in crush width made precise counts of cellsdifficult, but similar crushes had indistinguisihhable-accumui-lations. In addition, qualitatively similar accumulations at thecrush were observed whether or not the nerve cord had been

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FIG. 1. In nerve cords stained with Hoechst 33258 dye andbathed in Mg2+, microglia accumulated at lesions. (Upper) Connec-tives were stained 25 min with Hoechst dye, crushed, cultured 24 hrin L-15 medium containing 23 mM Mg2l, fixed, and Feulgen-stained.(Lower) Control connectives were not stained with Hoechst dye butwere crushed, cultured 24 hr in L-15 with no additional Mg2+, fixed,and Feulgen-stained. (Bar = 20 j.&m.)exposed to the viewing light used during the tracking exper-iments described below.

Tracking Microglia in Living Nerve Cords. Microglia beganmoving toward the crush within minutes after a lesion wasmade. Accumulation of microglia was seen 2 hr after crushingand increased, reaching peak levels within 24 hr. An exampleof sequential views of the connectives, photographed at 5-mmnintervals, is shown in Fig. 2. To assist in detecting movingcells, the image processor was used to subtract each imagefrom the next recorded 5 min later, as shown in the bottom sixpanels of Fig. 2. Those portions of the image that changedappear black (before) and white (after). Continuous recordingson videotape confirmed the identity of particular cells.Only a fraction of cells moved at any time, and those cells

moved at various speeds. For example, cell b moved towardthe crush at a nearly steady speed from 15 min to 35 min, asindicated by the lengths of the arrows between the black andwhite images in the middle four panels of Fig. 2. After 35 min(panel 40' -35'), cell b paused but then began to move again(panel 45'-40'). In contrast, cell a moved at different speedsduring the 25-mmn period of observation shown (note thevaried lengths of arrows between the before and after imagesfor cell a in Fig. 2).Some of the moving cells were moving within the viewing

window at the start of the 2- to 6-hr observation period (cellsa and b), others moved into the field of view and on towardthe crush (cell c), and some that had been initially stationarybegan moving while under observation. Some microgliamoved continuously; others stopped and started or occasion-ally reversed direction. Movement away from the crush wastemporary, never exceeded the average dimensions of themicroglia cell (see below and Fig. 3C), and in some casesaccompanied a rare movement laterally within the connec-tive. Few cells disappeared from the shallow plane of focus,

Proc. Natl. Acad. Sci. USA 86 (1989)

Neurobiology: McGlade-McCulloh et al.

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FIG. 2. Migration of microglia within the living nerve cord. Panels are views at 5-min intervals of a 0.2-mm length of connectives crushed -0.2mm to the right ofthe region viewed. In two top panels, photographed 15 and 20 min after crushing, microglial and perineurial sheath nuclei are visibleas bright fluorescent spots. Most ofthose microglia that moved traveled to the right, toward the crush (e.g., cells a and b). To highlight moving cells,the image recorded at one time (e.g., 15 min) was subtracted from that recorded 5 min later (20 min), creating a difference image (20' - 15'; thirdpanel). In the difference image, the earlier positions of cells appear as dark spots and the later positions are white. Cells that did not move do notappear in the difference image. The six bottom panels show difference images taken at successive 5-min intervals. Three ofthe moving microglia arelabeled a, b, and c, and their movements are marked with arrows. Although most cells of the perineurial sheath did not move, slight drift orjitter ofconnectives was in some cases detectable, as in the case of the cell marked with an asterisk.

the edges of the connectives and moved little. Shifting ofperineurial sheath cells thus served to indicate any gross

which would in effect have required lateral movement withinthe connectives. Nuclei ofcells ofthe perineurial sheath lined

1096 Neurobiology: McGlade-McCulloh et al.

movement of the nerve cord. In some experiments, theintensity of the excitation light was briefly increased 3-foldevery 20 min to improve image quality and to determinewhether additional stained cells were present but undetectedwith dim illumination. When such cells were seen, they were

out of the plane of focus.In nerve cords that had not been crushed, microglia did not

move. These controls were prepared as experimentals: theday before observation they were stained with Hoechst dyeand connective segments were pinned straight. Two controlsegments were each observed for 2 hr under the usual dimillumination. No nucleus moved perceptibly relative to othersin the connective, which included stationary cells of theperineurial sheath. The limit of detection in these experi-ments was a relative movement of 5 ,Am in 2 hr.

Microglia that did move within crushed connectives wereclocked at speeds up to 7 ,um/min measured over a 10-mininterval (Fig. 3A). The average speed during periods ofmovement was 1.9 Aum/min with a range up to 4.9 ,um/min(Fig. 3B). Measurements were made at 10-min intervals for 56cells in 10 preparations. The percentage of microglia movingat any time during the observation period varied as cellsentered and left the field of view (see, e.g., cells c and a in Fig.2) and varied from preparation to preparation, but typicalvalues ranged from 15% to 40% of the microglia. The rest ofthe microglia did not move during observations up to 6 hr;movements >5 pAm (i.e., <1 pum/hr) would have beendetected. Cell movement en masse at <1 pum/hr could nothave produced the accumulation seen in this and earlierstudies (9). A speed more than an order of magnitude fasterwould have been required. Movement away from the crush,indicated by a negative number, did not exceed the average

dimensions of the microglial cell (Fig. 3C; see below). Therange of distances traveled by individual nuclei duringperiods of movement was -41 pum to >200 pAm. Somemicroglial cells entered, traversed, and left the field of viewduring the period of observation. These cells traveled fartherthan the 220-,um length of connective under observation.

In one case, no MgCI2 was added and the preparation was

followed for 2 hr. Only a fraction of the microglia moved,traveling at speeds that were entirely within the range ofthose in elevated [Mg2"]. Muscular contractions of theconnectives made analysis of the shifting nerve cord labori-ous but confirmed that the addition of Mg2" did not measur-

ably affect microglial migration within the nerve cord, a

conclusion first based on Feulgen-stained preparations.Microglial Dimensions and Microglial Movements. Because

Hoechst dyes stain only nuclei and thus whole cells cannot beseen, any nuclear movements that are smaller than thedimensions of the cell could be nuclear movements within a

stationary cell. It was therefore important to determinewhether whole cells were moving. Measurements were madeof 18 randomly selected whole microglia in preparationsstained with silver carbonate to mark microglial cytoplasm.Their average length was 57 + 17pum (mean ± SD) and theirwidth was <5pum. Microglial nuclei were situated 40% ± 7%(mean ± SD) of the distance from an end of the cell, indicatingthat almost all nuclei lay within the central one-third of thecell. Cells that traveled backward all fell within this range; themaximum distance was -41 pum (Fig. 3C). Of course, thosenuclei that traveled forward <41 pum could be similarlyaccounted for as nuclear movement within a stationary cell,but more than two-thirds of moving nuclei traveled fartherthan this toward the crush and most moved more than thetypical cell's dimensions.The same results can be used in considering a converse

proposition, that the cell could move within the limits of itsdimensions without shifting of the nucleus. This could in

theory occur for a few cells, but it would not account for thelarge accumulation of microglia at the crush.

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FIG. 3. Distributions of velocities and distances traveled bymoving cells. Fifty-six moving microglia were tracked at 10-minintervals in 10 preparations. Velocity was computed from thedistance traveled during each interval and an average velocity of eachcell was determined for all intervals during which the cell moved. (A)The peak velocity was binned at intervals of 1 Aum/min. (B)Distribution of average velocities of the cells, averaged over 10-minintervals in which they moved, not necessarily the total observationperiod. (C) The distance traveled is shown for each cell, representedby a circle. The length of connective under observation was always220 Am; thus, only those cells that traversed the field of view wererecorded as traveling as much as 220 t.m. Cells initially in the fieldthat exited toward the crush may have traveled farther than indi-cated, but no cells moving away from the crush entered or left thefield of view.

DISCUSSIONMorgese et al. (9) showed that microglia migrate within theleech nerve cord to crushes and do so within 24 hr of injury.

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Proc. Natl. Acad Sci. USA 86 (1989)

Proc. Natl. Acad. Sci. USA 86 (1989) 1097

Their work raised questions about the route, velocities, andpopulation of cells moving. The present study using low-lightvideo microscopy confims the earlier results and answers

several of these questions.Microglia near the lesion begin moving almost immediately

toward it, but only a fraction (15-40%o) of the microglia moveat any time, and they move at different speeds. Thus, the cellsdo not move en masse. The microglia travel directly down theconnectives, apparently along axons, rather than movinglaterally to the edge of the connectives, beneath the perineu-rial sheath, and then along the nerve cord to the lesion.Lateral movements, which would also have been detected as

the spontaneous appearance or disappearance of cells fromthe focal plane, were rare. It is interesting that not allmicroglia moved at any time, because with suitable multiplecrushes essentially all the microglia within a segment ofconnective can move to lesions within 1 day (9). Why onlysome cells respond at any one time to the stimulus and whatdetermines their speeds remain unknown.

In mammals and other vertebrates, there may be severalsources for microglia, including those that arise endoge-nously within the brain and not from blood macrophages (1).It has not been possible to track microglial migration withinthe nervous system in vivo, but in cultures of dissociatedbrain cells, microglia are motile (3). While vertebrate micro-glia may divide, there is no evidence that the accumulation ofmicroglia in the leech is due to cell division (K.J.M.,unpublished results).

Vertebrate and leech microglia are phagocytic and removecellular debris after nerve injury (16, 18), but their functionsduring regeneration might extend farther. For example,similar macrophages have been proposed to provide regen-

erating axons with lipid and perhaps other molecules (19).When isolated leech neurons are plated in tissue culture, it

is common for microglia to accompany the neurons andmigrate onto the culture dish. Chiquet and Nicholls (20)observed that microglia seem to block neurite outgrowth andthat growing neurites avoid the microglia, circumnavigatingthem. The apparent inhibition of axon growth may relate toan ability of the microglia to channel or direct the growth ofaxons through the lesion. To judge from their elongated

nuclei, microglia within the lesion are oriented along the axisof the nerve, and they move into the lesion long before theregenerating axons. Perhaps microglia can also move to makeway for axons traversing the lesion.

We thank Xiaonan Gu, David McCulloh, and Steve Young forvaluable discussions and Bob Keane for critical reading of themanuscript. This work was supported in part by National Institutesof Health Grants RO1-NS20607 to K.J.M., F32-NS07488 to E.M.-M., and 5T32-NS07044.

1. Ling, E. A. (1981) in Advances in Cellular Neurobiology, eds.Fedoroff, S. & Hertz, L. (Academic, New York), Vol. 2, pp.33-81.

2. Giulian, D. & Baker, T. J. (1985) J. Cell Biol. 101, 2411-2415.3. Giulian, D. & Baker, T. J. (1986) J. Neurosci. 6, 2163-2178.4. Smith, P. J. S., Howes, E. A. & Treherne, J. E. (1987) J. Exp.

Biol. 132, 59-78.5. Hickey, W. F. & Kimura, H. (1988) Science 239, 290-292.6. Nicholls, J. G. (1987) The Search for Connections: Studies of

Regeneration in the Nervous System of the Leech (Sinauer,Sunderland, MA).

7. Coggeshall, R. E. & Fawcett, D. W. (1964) J. Neurophysiol.27, 229-289.

8. Kai-Kai, M. A. & Pentreath, V. W. (1981) J. Comp. Neurol.202, 193-210.

9. Morgese, V. J., Elliott, E. J. & Muller, K. J. (1983) Brain Res.272, 166-170.

10. Streit, W. J. & Kreutzberg, G. W. (1987) J. Neurocytol. 16,249-260.

11. Macagno, E. R. (1980) J. Comp. Neurol. 190, 283-302.12. Nicholls, J. G. & Baylor, D. A. (1968) J. Neurophysiol. 31,

740-756.13. Ready, D. F. & Nicholls, J. (1979) Nature (London) 281,67-69.14. Wallace, B. G., Adal, M. N. & Nicholls, J. G. (1977) Proc. R.

Soc. London Ser. B 199, 567-585.15. Muller, K. J. & Carbonetto, S. (1979) J. Comp. Neurol. 185,

485-516.16. Elliott, E. J. & Muller, K. J. (1983) J. Neurosci. 3, 1994-2006.17. Humason, G. L. (1962) Animal Tissue Techniques (Freeman,

San Francisco), p. 203.18. Elliott, E. J. & Muller, K. J. (1981) Brain Res. 218, 99-113.19. Ignatius, M. J., Shooter, E. M., Pitas, R. E. & Mahley, R. W.

(1987) Science 236, 959-962.20. Chiquet, M. & Nicholls, J. G. (1987) J. Exp. Biol. 132, 191-206.

Neurobiology: McGlade-McCufloh et al.