JOURNAL OF NEUROTRAUMAVolume 16, Number 4, 1999Mary Ann Liebert, Inc.

Short Communication

a i-Tubulin Expression in Proximally Axotomized MouseCortical Neurons

E.J. ELLIOTT,12 D.A. PARKS,1 and P.S. FISHMAN12

ABSTRACT

This paper further characterizes the response to axotomy of mouse transcallosal cortical neurons,a population of neurons that seems to be particularly refractory to regeneration. Mouse transcal-losal cortical neurons did not upregulate mRNA for the growth-associated protein artubulin fol-lowing axotomy, even when the axonal distance from injury to cell body was only 100-300 /int. Pre-vious experiments had found no upregulation of another growth-associated protein, GAP-43, bytranscallosal neurons following axotomy 1-2 mm from the cell body. These latest results establishthat this population of neurons fails to respond to axotomy even when it is extremely proximal andthat this failure is not a peculiarity specific to one growth-associated protein but is indicative of a

generally poor regenerative response.

Key words: artubulin; axonal regeneration; axotomy; CNS; cortical neurons; injury

IINTRODUCTION in other regions of the CNS. In particular, the population

of cerebral cortical neurons that project to the spinal cord,N adult mammals, neurons that extend axons into the or corticospinal neurons, have been found not to upreg-periphery can regenerate those axons following axot- ulate tubulin isotypes when axotomy is several mm away

omy. This regeneration is associated with upregulation (Mikucki and Oblinger, 1991), nor to upregulate GAP-of growth-associated proteins, such as GAP-43 (Skene 43 unless the distance between cell body and injury siteand Willard, 1981; Hoffman, 1989) and specific isotypes is less than 300 pm (Tetzlaff et al., 1994).of tubulin (Miller et al., 1989; Bisby and Tetzlaff, 1992; We have studied a second population of neurons in theMoskowitz and Oblinger, 1995). Some central neurons cerebral cortex, transcallosal neurons, which are corticalwith axons that lie totally within the central nervous sys- pyramidal neurons that project across the corpus callo-tem (CNS) will also sprout and/or upregulate growth-as- sum to innervate the contralateral cortex. Our initial mor-sociated proteins following axotomy, if the lesions occur phologic study of this neuronal population found mini-close enough to the cell body (Doster et al., 1991; Tet- mal evidence of axonal sprouting (Fishman and Mattu,zlaff et al., 1991; McKerracher et al., 1993; Vaudano et 1993). That study also illustrated the difficulty in recog-al., 1995). However, the regenerative response of central nizing relatively few axons with morphologies consistentneurons varies, and neurons in the cerebral cortex have with regeneration among the large numbers of degener-been found to respond less readily to injury than neurons ating axons (greater than 90%), and motivated us to eval-

1 Research Service, Veterans Administration Medical Center, Baltimore, Maryland,department of Neurology, University of Maryland at Baltimore, Baltimore, Maryland.

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uate initial attempts at regeneration with more quantita-tive molecular techniques. Although that study found thelargest number of putative axonal sprouts at 10 days af-ter injury, our in situ hybridization studies have focusedon the first week after injury. The rationale for this changeis threefold: (1) Increase expression of regeneration re-

lated RNA generally precedes morphologic evidence ofregeneration. (2) Lesions in the current study design are

more proximal, and we anticipated that cellular changeswould occur at an accelerated pace. (3) Death becomesa prominent cellular response at later intervals after in-jury, further complicating interpretation of regenerationrelated changes. In previous work, we found that trans-callosal neurons in the mouse did not upregulate GAP-43 mRNA following axotomy 1-2 mm from the cell body(Elliott et al, 1997), consistent with the minimal axonalsprouting seen after injury. In this study, we have soughtto determine whether mouse transcallosal neurons fail to

respond to injury that is extremely proximal (only a fewhundred microns away from the cell body), and whetherthe failure extends generally to other growth-associatedproteins besides GAP-43. The ai isotype of tubulin (de-noted Tai in rats and Mai in mice) appears to be regu-lated independently from GAP-43 (compared Gloster etal, 1994, and Nedivi et al, 1992), and its response to in-jury can differ from that of GAP-43 (Tetzlaff et al, 1994;Kittlerova et al, 1996; Fournier et al, 1997). Thus, we

have performed in situ hybridization with an Mai probeto see whether mouse transcallosal neurons respond toclose axotomy with changes in Mai tubulin mRNA.Moreover, we have examined transcallosal neurons verynear to the wound and deep in the cortex, for which theproximal axon stump after axotomy is only 100-300 pmlong. This corresponds to an actual distance of the cellbody away from the wound site as little as 25-50 pm.

MATERIALS AND METHODS

Adult C57/B1 6 mice (10 weeks of age or older, fromJackson Laboratories) were used in these experiments.Cortical pyramidal neurons that project across the corpuscallosum were labeled by retrograde uptake of the fluo-rescent dye Fluoro-Gold, as described previously (Elliottet al, 1997). This procedure involves placing a dyesoaked gelatin sponge over the fronto-parietal cortex ofone hemisphere, and labels a large number of transcal-losally projecting neurons that reside in the homotypiccontralateral cortex (Fig. 1). A week after labeling, a

knife wound was made through the cortex and underly-ing fiber tracts, positioned to divide the retrogradely la-beled cortical neurons into two populations: (1) Fluoro-Gold-labeled neurons lateral to the wound were

axotomized near the cell body, while (2) labeled neurons

medial to the wound were left intact. The labeled neu-

rons medial to the knife wound (unaxotomized) were de-signed to serve as a control populations.

Animals were sacrificed at 1 day, 3 days, and 7 daysafter injury. Brains were treated as described previously(Elliott et al, 1997). Briefly, they were perfusion-fixed,removed and postfixed for 4 h, incubated overnight in30% sucrose to provide cryoprotection, frozen on dry iceand cryosectioned at 14 pm. Although this protocol gavea higher background with in situ hybridization than woulda procedure with little or no fixation, this amount of fix-ation was necessary to stabilize the Fluoro-Gold suffi-ciently to withstand the dehydration and other treatmentsof the tissue and the extensive rinsing involved in the insitu hybridization protocol. All cells evaluated for in situhybridization appeared viable without morphologic signsof apoptosis or necrosis. Ability to retrogradely transportand retain Fluoro-Gold within the soma and dendrites was

also taken as evidence of neuronal viability.Sections were hybridized overnight with a 33P-labeled

oligonucleotide probe for mouse Mai tubulin mRNA,taken from the 3' noncoding region of the gene [nu-cleotides 594-632 (Lewis et al, 1985)], which is the re-

gion in which the various isotypes oftubulin differ (Lewiset al, 1985). Slides were rinsed 1-2 h in 0.5 X SSC at52°C, dipped in NTB-2 emulsion, and allowed to exposefor 4-8 days at 4°C in the dark. After development of theslides, computer-based image analysis software by Sig-nal Analytics Corporation was used to capture and su-

perimpose images of fluorescent cells and silver grains,and to measure the area of cells and silver grains. As a

positive control, sections of mouse lumbar spinal cordcontaining axotomized motor neurons were included inthe in situ hybridization.

RESULTS

In the rat, axotomized spinal motor neurons regener-ate vigorously and upregulate Tai mRNA (Miller et al,1989). In our experiments, axotomized mouse motor neu-

ron also showed clear upregulation of Mai tubulinmRNA (Fig. 2A,B). The signal was absent in excess-coldcontrols, i.e., in sections on the same slide which were

incubated in hybridization medium containing 100-foldexcess cold probe in addition to the radioactively labeledprobe.

In contrast to these positive controls with spinal mo-

tor neurons, there was no obvious silver grain clusteringin the cortex over the Fluoro-Gold-labeled cell bodies ofaxotomized transcallosal neurons (Fig. 2C,D). Comput-erized counts of silver grains over axotomized and un-

JJ^f

ai-TUBULIN IN AXOTOMIZED CORTICAL NEURONS

FIG. 1. Schematic of surgical procedures for neuronal labeling and lesioning. (A) A gelatin foam sponge saturated withFlouro-Gold resting on the surface of the frontoparietal cortex following a large craniotomy. Cortical neurons projecting acrossthe corpus callosum are labeled by retrograde axonal transport of dye. Most of these cells are pyramidal neurons located incortical regions homotopic to the contralateral label source. (B) The location of a knife wound performed 7 days after the ini-tial labeling. At that time, the gelfoam sponge is devoid of dye and is removed. The knife wound is made close to perpen-dicular to the cortical surface in an attempt to divide the labeled field of transcallosal cell into a medial group (unaxotomizedcontrol cells) with uninjured axons still projecting to the contralateral hemisphere, and a lateral group of cells that have beenaxotomized by the wound.

--

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FIG. 2. (A) Fluorescent light micrograph of a spinal cord cross section shows the Fluoro-Gold-labeled cell bodies of mo-

tor neurons that were axotomized 7 days earlier by sciatic nerve crush. (B) Darkfield light micrograph of the same field ofview as in (A) shows silver grain clustering over the spinal motor neuron cell bodies, following in situ hybridization with theprobe for Mai tubulin. Silver grains are obviously clustered over axotomized motor neurons, which has a silver grain density13 times that of nearby background. (C) Fluorescent light micrograph of a section through parietal cortex shows the Fluoro-Gold-labeled cell bodies of transcallosal pyramidal neurons that have been axotomized 3 days earlier. (D) Darkfield light mi-crograph of the same field of view as in (C) shows the absence of silver grain clustering over axotomized transcallosal cellbodies. Bar = 20 pm.

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ai-TUBULIN IN AXOTOMIZED CORTICAL NEURONS

Neurons 0.5 - 2.0 mmfrom axotomy site

B Neurons 100 - 300 Limfrom axotomy site

13 5 7

Days after injury13 5 7

Days after injuryFIG. 3. Comparison of a i -tubulin expression by axotomized neurons with that of unaxotomized neurons. The axotomized/un-axotomized ratios of mean silver grain counts/neuron are plotted for cortical neurons axotomized within 0.5-2 mm of the cellbody (A) and for cortical neurons axotomized within 100-300 pm of the cell body (B). Each point represents one animal. Theanimals used for B are a subset of the animals used for A, but the neuronal populations differ. The means in A are of 30-40 ax-

otomized and 30-40 unaxotomized neurons, while the means in B are of 10 neurons each.

axotomized neurons, and statistical comparison of thetwo means, confirmed this visual impression. For eachof 22 mice, silver grains were counted over 30-40 axo-

tomized transcallosal neurons and over 30-40 unaxo-tomized transcallosal neurons, within 0.3-2 mm of thewound. For each experimental animal, the two means

were compared for statistically significant difference bythe two-tailed Student's t test. The difference was statis-tically significant (p < 0.01) in half the animals. How-ever, even in these statistically significant cases, the dif-ferences were not greater than those typically seen

between transcallosal neurons of two randomly chosencortical regions in an unwounded animal. Moreover, insome of the cases of statistically significant difference, itwas the mean axotomized grain count that was greater,while in others it was the mean unaxotomized grain countthat was greater. Thus, the differences in axotomized andunaxotomized signal were judged not to be biologicallysignificant, especially when compared with a spinal mo-

tor neuron signal of X13 background. These results are

illustrated in the scatter plot in Fig. 3A, which shows theratio between the mean grain count for axotomized neu-

rons and the mean grain count for unaxotomized neurons,for the 22 animals studied.

Eleven of the 22 experimental animals had Fluoro-Gold-labeled neuron cell bodies that were very close bothto the fiber tract and to the wound, with estimated axon

lengths from cell body to axotomy site of 100-300 pm.In each of these 11 animals, the mean silver grain countwas determined for 10 extremely proximal, axotomizedneurons and 10 unaxotomized neurons equally proximalto the wound. The axotomized/unaxotomized grain countratio varied from 0.75 to 1.55 for these 11 animals (Fig.3B). In none of these cases was the difference in themeans of the two populations statistically significant (p <

0.01). Thus, for these extremely close neurons, the re-

sults were less variable than for the larger population ofneurons farther from the wound, and indicated even more

compellingly that there was no meaningful difference inMai tubulin mRNA levels for axotomized neurons com-

pared with unaxotomized neurons.Trauma-associated effects of the wound, such as in-

flammation or ischemia, might cause neurons very near

the wound, whether axotomized or not, to respond by up-regulating growth-associated proteins (Lu and Richard-son, 1995). If so, then Mai tubulin expression in axo-

tomized neurons might be underestimated by comparingit with near-by unaxotomized neurons. To address this

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question, the value of mean silver grains/cell for unaxo-tomized transcallosal neurons very near the wound was

compared with the mean silver grains/cell for a distantcontrol population of cortical neurons in the contralateraltemporal cortex. Proximal/distant ratios were determinedfor the 11 experimental animals with extremely proximalneurons and for four animals with no wound in the pari-etal cortex, but with Fluoro-Gold labeling of cortical neu-

rons. The ratios showed a similar range in values for thewounded (0.4-1.0) and unwounded (0.4-1.8) animals.

DISCUSSION

These results indicate that, in wounded animals, un-

axotomized transcallosal neurons very near the wounddid not upregulate Mai tubulin, and comparing axo-tomized neurons with them did not underestimate possi-ble axotomy-induced increases. Thus, transcallosal cor-tical neurons in the mouse did not upregulate thegrowth-associated protein Mai tubulin following even

very proximal axotomy, 100-300 pm from the cell body.For reasons that are not yet clear, central neurons fromdifferent regions of the brain vary in the minimal dis-tance from the cell body within which axotomy must oc-

cur before it stimulates upregulation of growth associatedproteins. In the case of rat retinal ganglion cells, the min-imal distance seems to be about 3 mm (Doster et al.,1991). In the case of rat corticospinal neurons, the min-imal distance has been reported to be about 200 pm (Tet-zlaff et al., 1994). For transcallosal cortical neurons inthe mouse, the minimal distance, if there is one, must beless than 100 pm.

One hypothesis concerning the relationship of prox-imity of axotomy to the cell body and initiation of re-

generation relates to the role of axon collaterals. Manytranscallosal neurons have extensive axon collateralswhich may be spared by axotomy even relatively closeto the cell body (Hartenstein and Innocenti, 1981). Thesecollaterals may provide a source of a target derived fac-tor that suppresses axonal regeneration. Target derivedneurotrophic factor may also be involved in cell death oftranscallosal neurons after axotomy. Although the neu-

rons evaluated in this study appeared viable (both on mor-

phologic grounds and by their retention of retrogradelytransported Fluoro-Gold) most are destined to die over

the next days and weeks (Fishman and Parks, 1998). Ul-traclose axotomy of a related neuronal population, corti-cospinal neurons by intracortical undercutting lesionsalso leads to dramatic cell loss, which can be reduced byinfusion of ciliary neurotrophic factors (Dale et al., 1995).The role of neuronal connectivity in regulating regener-ative changes and/or cell death after axotomy is also sup-

ported by developmental studies of corticospinal neurons.

Synthesis of both GAP 43 and tubulin fall to low adultlevels during the neonatal period, a time where wide-spread connections in both brain and spinal cord are

formed (Kalil and Skene, 1986). After the neonatal pe-riod, axotomy at the level of the medullary pyramids isno longer associated with either induction of growth as-

sociated protein expression or axonal regeneration (Rehet al., 1987). Pyramidal lesions also result in extensivecell death of corticospinal neurons in neonatals, but notin adult animals (Merline and Kalil, 1990). Collaterals ofthe proximal axon spared by a lesion may provide a

source of a target derived factor that maintains neuronalintegrity but suppresses axonal regeneration.

The role of connectivity is not supported by studies ofretinal ganglion cell regeneration where differences are

seen in vigor of regenerative changes with more proxi-mal optic nerve lesion even though both proximal anddistal lesions disrupt ganglion cell connectivity to an

equal extent (Dorster et al., 1991). These observationssuggest a role for loss of axoplasm or wound related ionicchanges as a signal for regeneration that may be magni-fied by more proximal lesions.

These results reveal mouse cortical neurons to give theleast regenerative response of any of the animal model sys-tems yet characterized, and to be an exception to the con-

cept of abortive central regeneration, first described by Ca-jal (Ramon y Cajal, 1959), in which there is an initialattempt at axonal elongation, but in which the response isterminated within a few days or weeks. Thus, the poorlyregenerating transcallosal mouse cells may be good can-

didates for investigating factors which limit the initial re-

sponse of central neurons to injury, and for investigatingwhat conditions might optimize regeneration in the humanbrain. Future experiments will focus on whether mousetranscallosal neurons can be stimulated by pharmacologi-cal interventions to respond regeneratively to injury.

ACKNOWLEDGMENTS

This work was supported by a Veterans Administra-tion Merit Review grant to P.S.F.

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Address reprint requests to:P.S. Fishman

Research Service, #151VA Medical Center

10 N. Greene St.Baltimore, MD 21201

e-mail: pfishman@umaryland.edu

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