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Effect of proximal axotomy on GAP-43 expression in cortical neurons in the mouse
Brain Research 755 Ž1997. 221–228
Research report
Effect of proximal axotomy on GAP-43 expression in cortical neurons in the mouse E.J. Elliott b
a,b,)
, D.A. Parks
a,b
, P.S. Fishman
a,b
a Research SerÕice, VA Medical Center, 10 N. Greene Street, Baltimore, MD 21201, USA Department of Neurology, UniÕersity of Maryland at Baltimore, Baltimore, MD 21201, USA
Accepted 30 December 1996
Abstract As an approach to understanding why central neurons fail to regenerate, we have studied the response to proximal axotomy of transcallosal neurons of the cerebral cortex of the mouse. Anatomical studies have indicated only very slight regenerative responses by this population of cortical neurons. To further examine the regenerative response of these cells, we have looked by in situ hybridization at the expression of GAP-43 mRNA following axotomy caused by a stab wound delivered within about 200 mm to 1.25 mm of the cell body. Axotomized transcallosal neurons were compared with near-by unaxotomized transcallosal neurons, as well as with distant unaxotomized cortical neurons in the contralateral hemisphere. All three populations of neurons had been pre-labeled with Fluoro-Gold to allow identification. No up-regulation of GAP-43 mRNA above background levels was detected for axotomized cortical neurons at 1, 3 or 7 days after injury. In contrast, increases in mean silver grain density of up to 8-fold were measured in axotomized spinal cord motor neurons used as positive controls. Thus, as a population, the transcallosal cortical pyramidal neurons did not show a significant regenerative response, as monitored by GAP-43 upregulation, even with very close axotomy. These results identify this population of neurons as among the least regenerative studied, and suggest that, on a molecular level, inherent neuronal properties play a role in the limited regenerative response to brain injury. Keywords: Axonal regeneration; GAP-43; Axotomy; Injury; CNS; Cortical neuron
1. Introduction Neurons completely within the central nervous system Žinstrinsic neurons. of adult mammals fail to regenerate severed axons, while neurons with axons in the periphery Žextrinsic neurons. generally sprout after axotomy and grow new axons w29x. Both local environmental factors and inherent neuronal characteristics contribute to this difference w4,6,12x. Changing the local environment of intrinsic neurons by grafting embryonic or peripheral nerve tissue into the CNS can result in sprouting and axonal growth by the intrinsic neurons into the graft w1,15x. However, neurons in different regions of the CNS show different capacities for growth into transplants w1,15,27x. For example, neurons in phylogenetically newer regions of the brain such as the neocortex have generally shown less regenerative response than those in other regions w4x. One measure of the inherent capacity for regeneration is
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Corresponding author. Fax: q1 Ž410. 605-7906.
the extent to which axotomized neurons respond by upregulating genes and proteins associated with axonal growth, in particular, the growth associated-protein GAP-43. Axotomy of extrinsic neurons is followed by several- to many-fold increases in levels of GAP-43 mRNA or protein w18,34x. Early experiments found that intrinsic neurons failed to upregulate GAP-43 after axotomy w20,30,34x, but more recently it has been demonstrated that a number of intrinsic neurons will both sprout and upregulate GAP-43 and other growth-associated proteins, albeit transiently in some cases, if the lesion occurs close enough to the cell body. Examples include retinal ganglion cells following proximal axotomy of the optic nerve w10x, rubrospinal motor neurons in the midbrain following cervical spinal lesions w35x, and thalamic neurons after a near-by thalamic stab wound w38x. In working toward the goal of understanding why intrinsic neurons fail to regenerate and discovering how they can be stimulated to regenerate, it can be instructive to study the best possible examples of regenerative failure, such as neurons of the cerebral cortex w4x. We have chosen
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 1 0 0 - 5
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to study transcallosal pyramidal neurons, a subpopulation of cortical neurons which project axons across the corpus callosum to the contralateral cerebral cortex. This population offers the opportunity for close axotomy Žby a stab wound. with relatively little disruption of the surrounding tissue, as well as the possibility for a control population of near-by, unaxotomized transcallosal neurons. This population also has many cellular features that are shared with cortical pyramidal neurons in higher species, including humans, where axotomy due to brain trauma or stroke has devastating clinical consequences. Anatomical studies of transcallosal cortical neurons have provided evidence for only a very slight regenerative response after axotomy in the hemisphere contralateral to the cell body, with only modest axonal sprouting amidst a preponderance of axonal degeneration w16x. We wished to further examine the regenerative response of these cells by looking at expression of GAP-43 after more proximal axotomy, especially in light of a recent report of upregulation of GAP-43 in another subpopulation of cortical pyramidal neurons, corticospinal neurons, following extremely proximal axotomy w14,36x.
2. Materials and methods 2.1. Animal surgery Adult male mice, C57rBl6 strain, 2–3 months old, from Charles River Labs, were used in all experiments. Mice were anesthetized by methoxyflurane inhalation before and during all surgeries. After surgery, mice received an intramuscular injection of the analgesic butorphanol tartrate, 0.1 ml of 0.1 mgrml. The general strategy was to label transcallosal pyramidal neurons so that they could be positively identified later in coronal sections of the brain, and then to deliver a knife wound that would axotomize some of these pre-labelled neurons, and leave others intact. Fig. 1 illustrates the labelling and injury procedures. Transcallosal pyramidal neurons in the right parietal cortex were labeled with the fluorescent dye Fluoro-Gold, which is taken up by axons and transported retrogradely to the cell body w32x. A gelfoam sponge saturated with 4% Fluoro-Gold in distilled water was placed, after a large Ž3 = 4 mm. craniectomy, on the surface of the left parietal cortex. The gelfoam was held in place by tacking it to the cranium with cyanoacrylate glue, and the overlying skin was sutured closed. This procedure resulted in homotopic labeling of a large number of transcallosal neurons in the contralateral cortex, as well as labeling of numerous cortical neurons in the ipsilateral cortex. One week after Fluoro-Gold labeling, a second surgery was performed in which the fiber tracts underlying the parietal cortex were completely transected by a 5 mm long knife wound through the right parietal cortex, 3 mm to the
Fig. 1. This line drawing of a coronal brain section illustrates the labeling of transcallosal cortical neurons with Fluoro-Gold ŽFG. applied to the contralateral surface of the brain in a gelfoam planchet, and the severing of axons of the most laterally located of those neurons with a knife wound.
right of the midline. This lesion typically was located within a large field of pre-labeled neurons. Neurons lateral to the lesion had their axons severed by it Ži.e. were axotomized., while neurons located medial to the lesion remained connected to their targets in the contralateral hemisphere, and served as unaxotomized controls. Although the axotomized pyramidal cell bodies were located not more than 100 to 500 mm from the site of the stab wound, the axotomy produced proximal axons which varied in length from about 200 mm to as much as 1.25 mm, depending on the exact location of the cell body in the cortex. This variability arises because the transcallosal axons travel ventrally down through the cortex to reach the subcortical fiber tracts and then turn toward the midline. Thus, more superficial cell bodies, and more laterally placed cell bodies, had longer segments of proximal axon remaining after the stab wound than did deeper or more medial cell bodies. 2.2. Histologic procedures Animals were sacrificed by anesthetic overdose at 1, 3 or 7 days after the knife wound. These time points were chosen after consideration of the time course of GAP-43 mRNA upregulation reported in other regenerating systems, in which maximal GAP-43 mRNA upregulation occurs from one day to one week after injury w8,10,22,35x. The chest was opened, a catheter inserted into the aorta via the left cardiac ventricle and the vascular system was perfused with 25 ml of cold 10% sucrose-PBS, followed by 25 ml of 10% formalin, 10% sucrose-PBS. Brains were removed and postfixed for 4 h at 48C in 10% formalin-30% sucrose-PBS. Postfixation was found to be necessary to stabilize the Fluoro-Gold sufficiently that it would be retained throughout the lengthy incubations and rinsings of the in situ hybridization ŽISH. procedure. Even so, there was considerable loss of fluorescence.
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Brains were frozen on dry ice immediately after the 4 h post-fix, or were immersed overnight in 30% sucrose-PBS, for additional cryoprotection, before being frozen on dry ice. Coronal sections Ž14 mm. were cut on a cryostat, and thaw-mounted onto Fisher Superfrost coated slides. Every fifth slide was examined to verify that the wound completely transected the fiber tracts and that there were Fluoro-Gold-labelled neuronal cell bodies both medial and lateral to the wound. The remaining slides were stored at y808C until use. For use as positive controls for sensitivity of detection of GAP-43 mRNA in the ISH experiments, motor neurons in the spinal cord were axotomized by sciatic nerve crush. At the time of the crush, 2 ml of a 4% solution of Fluoro-Gold was injected inside the perineurial sheath and proximal to the lesion, using a hand-held Hamilton syringe. One week later, animals were perfused and the lumbar spinal cord was removed, post-fixed, cryoprotected and sectioned. Sections containing Fluoro-Gold-labelled neuron cell bodies were used in ISH experiments. Unlabelled, unaxotomized motor neurons on the contralateral side of the same sections were made visible by counter staining with Cresyl violet and were recognizable by their large size and characteristic position. 2.3. In situ hybridization Frozen brain sections and spinal cord sections, sealed in boxes with silica gel dessicant, were thawed at room temperature. Thawed sections were treated with proteinase K Ž5 mgrml in PBS. at 378C for 4 min, acetylated with 0.25% acetic anhydride in 0.1 M TEA, dehydrated through graded ethanols, delipidated in chloroform, and allowed to air dry. Dried sections were circled with a PAP pen ŽResearch Products International Corp., Mt. Prospect, Illinois.. The resulting outline of hydrophobic residue around each section allowed the two sections on each slide to be hybridized to 50 ml droplets of two different solutions- one containing 33P-labelled GAP-43 probe and a second containing 1600-fold excess unlabelled probe in addition to the labelled probe. This provided a negative control on each slide for specificity of probe binding. Any signal that remained in the ‘excess-cold’ section was considered nonGAP-43-specific. All sections were incubated 1 h at room temperature in a prehybridization mixture Ž47% formamide, 10% dextran, 4 = SSC buffer w150 mM NaCl, 15 mM sodium citrate, pH 7.0x, 1 = Denhardt’s reagent, 0.25 mgrml yeast tRNA, 0.5 mgrml sheared and boiled herring sperm DNA, 10 mM Tris, 1 mM EDTA. which contained no probe but did contain poly dA Ž5 mgrml, Boehringer-Mannheim Corporation, Indianapolis, IN, USA.. The sections were then incubated overnight at 378C in humidified chambers in the above hybridization medium containing 5 = 10 7 cpmrml Ž0.5 pmolrml. of 33 P-labelled GAP-43 probe, or contain-
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ing 33 P-labelled probe plus 800 pmolrml of unlabelled GAP-43 probe. The GAP-43 antisense probe sequence was complementary to bases 210–248 of mouse GAP-43 mRNA w9x. The oligonucleotide was synthesized and purified by the Biopolymer Laboratory of the University of Maryland at Baltimore, and labelled at the 3X-end using terminal deoxynucleotidyl transferase ŽBoerhinger-Mannheim. and walpha- 33 PxdATP ŽNew England Nuclear, Boston, MA, USA. to a specific activity of 1–3 =10 9 c.p.m.r mg. After overnight incubation, the slides were rinsed in 1 = SSC at room temperature, four changes of 0.5 = SSC at 528C in a shaking water bath, and two changes of 0.5 = SSC at room temperature. Sections were dehydrated through ethanols, air dried and dipped in liquid emulsion ŽNTB2 nuclear track emulsion, Kodak, Rochester, NY, USA. diluted 1:1 with sterile water. Emulsion-coated slides were exposed for 4–11 days in the dark at 48C, developed in Kodak D-19 developer Ždiluted 1:1., dehydrated, cleared in xylene, and coverslipped with D.P.X. neutral mounting medium ŽAldrich Chemical Co., Milwaukee, WI, USA.. Sections were viewed both with epi-illumination through a UV filter set and with bright field illumination. Fluorescent images and bright field images were captured using a Cohu video camera, Scion frame grabber, and IP Lab Spectrum image analysis software by Signal Analytics ŽVienna, VA, USA.. Fields of view were chosen for image analysis which offered a high density of fluorescentlylabelled cells. Cells chosen for analysis were those with a relatively uniform size Ž8–12 mm long. and shape Žpyramidal to elliptical.. Silver grains over Fluoro-Gold-labelled cells were counted by superimposing fluorescent and bright field images. Cellular cross-sectional areas were computed from outlines that were hand-drawn around the fluorescent cell bodies. Within these cellular outlines, the area occupied by silver grains was measured by the computer and converted to a grain count by dividing the grains area by a mean grain size calculated by the software for that particular image. This grain count was expressed as an absolute number of grains per ‘cell profile’ Ži.e. grains per section of cell., and as a grain density. For each animal, grains over ; 30 axotomized cortical neurons and ; 30 control, non-axotomized neurons were counted on the same slide, and means were calculated and compared for significant difference using a two-tailed ttest. The ratio of the two means was determined for each slide, thus normalizing slide-to-slide variations in background signal. Eight to nine animals were examined at each time point. Background levels of silver grain density could not be quantitatively determined by measuring grain density from regions near the fluorescently labelled axotomized and non-axotomized neurons, because these regions might contain transcallosal cortical neurons which had not filled with Fluoro-Gold or which had lost the dye during the ISH protocol. Two different estimates of background grain
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density were obtained- one from Fluoro-Gold labelled neurons in the excess-cold sections, and a second from a population of Fluoro-Gold labelled neurons located in the temporal region of the contralateral cerebral cortex. This second population of neurons representated cortical pyramidal neurons distant from the wound. As a positive control for detection of GAP-43 mRNA, spinal cord sections containing axotomized motor neurons were included in the ISH experiments. It has been established that spinal motor neurons upregulate GAP-43 mRNA following sciatic nerve section w22x. The extent of upregulation in the spinal motor neuron controls was determined by comparing grain density over axotomized motor neurons with grain density over unaxotomized motor neurons on the contralateral side. The unaxotomized cell bodies, which were not fluorescently labeled, were visualized by light counterstaining with cresyl violet. In 3 out of 15 spinal motor neuron preparations that were examined,
GAP-43 upregulation, as well as faint Fluoro-Gold labelling, was observed in motor neurons contralateral to the axotomized motor neurons. Such a contralateral effect has been reported by others w22x and might be due to the presence of very fine collaterals that extend to the contralateral side. None of these animals were used as positive controls for ISH experiments.
3. Results After Fluoro-Gold application to the left parietal cortex, fluorescent transcallosal pyramidal neurons were found at depths corresponding to layers II, III and V of the right parietal cortex. Since sections were not stained to show cell bodies, it was not possible to assign cells unequivocally to a particular layer. However, these assignments agree with anatomical studies of rat callosal projections
Fig. 2. Light micrographs of coronal sections through the cerebral cortex of a mouse 1 day after a stab wound to the parietal region. Transcallosal neurons were pre-labelled with Fluoro-Gold 7 days before the wound. Unaxotomized cortical neurons, medial to the wound, are shown with epi-fluorescent illumination in ŽA., and the same field of view is shown with simultaneous brightfield and fluorescent in ŽB.. Axotomized cortical neurons, lateral to the wound, are shown in epi-fluorescent illumination in ŽC., and the same field of view is shown with simultaneous brightfield and fluorescent illumination in ŽD.. There is no obvious grain clustering over either unaxotomized or axotomized transcallosal neurons. Calibration bars s 20 mm.
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w19,40x. Fluoro-Gold-labelled neurons generally showed both diffuse, cytoplasmic-wide fluorescence and punctate vesicular fluorescence Žsee Fig. 2A.. Following a stab wound to the right parietal cortex, fluorescent pyramidal cells medial to the wound appeared intact and similar to labeled neurons in uninjured animals. Lateral to the wound, the number Žper unit area. of fluorescent cells was generally much less than that of cells medial to the wound. Moreover, the number of fluorescent lateral Žaxotomized. cells was generally smaller at 7 days following injury than at 1 day. This is consistent with our observation that many transcallosal neurons die following proximal axotomy. After in situ hybridization, there was no obvious grain clustering over axotomized transcallosal neurons, in any of the animals at any of the post-lesion times ŽFig. 2.. Thus it was clear that there was no upregulation of GAP-43 that approached the level seen in spinal cord motor neurons, for which grain clustering was obvious ŽFig. 3.. To look more closely for possible smaller changes in GAP-43 levels, mean silver grain counts were compared for axotomized and unaxotomized neurons in each of the 26 animals. The mean grain density for both axotomized and unaxotomized neurons was in the range of background grain density, as estimated from excess-cold sections and from cortical neurons distant to the wound. Thus, it was not possible to subtract this background value without arriving at negative values in many cases. When mean grain densities, uncorrected for background, were compared, axotomized cortical neurons did not differ significantly from unaxotomized cells in the majority of animals ŽFig. 4A.. This contrasted with the spinal cord control preparations, in which axotomized motor neurons showed from 2- to 8-fold increases in silver grain density compared to unaxotomized motor neurons. Seven of the 26 experimental animals Žindicated with
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asterisks in Fig. 4A. showed differences in grain density between axotomized and unaxotomized cortical neurons that were statistically significant Ž P - 0.0006.. However, these differences included both increases and decreases, and were modest in size compared with the positive controls. For example, for the cortical preparation with a 70% higher grain density in axotomized neurons, the matched spinal cord control gave an 8-fold higher grain density over axotomized motor neurons. Moreover, neither the magnitude nor direction of the differences correlated with probable length of proximal axon for the cells involved. Cell swelling is a well characterized part of the response of extrinsic neurons to axotomy w3x. If cell swelling occurred for the transcallosal cortical neurons in these experiments, then the amount of mRNA per unit volume Žor per unit cross-sectional area. would be diluted, and estimates of mRNA based on density would be underestimates. Fig. 4B shows a plot of the mean cross-sectional cellular area, expressed as the ratio of axotomized to unaxotomized cells, vs. time after injury, for the 26 animals studied. For most animals, the ratio was about 1.0, indicating no change in cell size. However, in 7 of the 26 animals, the difference in mean cross-sectional area between axotomized cells and unaxotomized cells was statistically significant Ž P - 0.003.. In all these cases, the mean axotomized cell area was 20 to 30% higher, suggesting cell swelling after axotomy. The animals with statistically significant differences are indicated with asterisks in Fig. 4B. Five of these seven were among the seven animals that showed changes Žeither increases or decreases. in silver grain density with axotomy. If cell swelling did occur, then actual grain counts per cell are a more accurate criterion for comparison than grain density. Fig. 4C shows the mean grain count per cell profile, for axotomized compared to unaxotomized cells. Grains per cell cross section are presented here, rather than
Fig. 3. Light micrographs of a cross-section of a spinal cord from a mouse 7 days after unilateral sciatic nerve crush. The section has been probed with the 33 P-labeled GAP-43 oligonucleotide probe and stained with cresyl violet. Axotomized spinal cord motor neurons Žin A. have an 8-fold greater density of silver grains than unaxotomized spinal cord motor neurons on the contralateral side Žin B.. Arrows point to examples of motor neuron cell bodies. Calibration bar is equal to 20 mm.
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grains per cell volume, because the nucleus was not visible in these unstained preparations and thus cytoplasmic volume could not be accurately estimated. The results based on grain countsrcell are similar to those based on grain density, that is, in most animals the grain counts are the same for axotomized and unaxotomized cells, though there are a few individuals with statistically significant differences, two with higher mean grain counts for axotomized cells and two with lower. The data from one of these individuals, with a 2-fold greater mean grain count for axotomized cells, is depicted in Fig. 5. This scatter plot of grain count vs. cellular cross-sectional area for individual Fig. 5. Scatter plot of grain count vs. cellular cross-sectional area for individual cortical neurons in an animal for which the mean grain count of axotomized neurons was 2-fold greater than that of unaxotomized neurons Žsame animal as illustrated in Fig. 2.. Time after axotomy s 7 days.
neurons shows that, in this animal, higher grain counts do not correlate with larger cross-sectional cellular area.
4. Discussion
Fig. 4. The ratio of the mean values for axotomized neurons compared to unaxotomized neurons, in each of the 26 animals studied, are plotted for: ŽA. silver grain density, ŽB. cellular cross-sectional area, and ŽC. silver grain count per cellular cross-scetion. Asterisks mark those animals for which the difference of the ratio from 1.0 was statistically significant Ž P - 0.0005 in A, P - 0.003 in B, P - 0.006 in C..
The GAP-43 measurements reported here extend previous morphological evidence that transcallosal neurons in the cerebral cortex show little regenerative response to axotomy w16x. That earlier work looked at transcallosal cells axotomized in the contralateral hemisphere, within 5 to 10 mm of the cell body. The work reported here looks at transcallosal cells axotomized in the ipsilateral hemisphere, within 200 mm to 1.25 mm of the cell body. These latest results show that, even with very proximal axotomy, there did not appear to be meaningful differences in GAP-43 mRNA expression between axotomized and unaxotomized transcallosal cortical neurons. In the few Ž4 out of 26. individuals for which there were statistically significant differences in mean grain counts for axotomized compared with unaxotomized neurons, the biological significance of the difference was not clear, since in all cases the grain counts were within the range of background levels, and in two cases the axotomized value was higher and in two cases it was lower. Of the two animals with higher axotomized grain counts, one was taken at 1 day after axotomy and the other at 7 days, and of the two animals with lower grain counts, one was at 1 day and the other at 7 days. Thus, there is no correlation with time after injury, and no ‘trend’ in the data, as might be seen if there were transient upregulation andror if there were a decline in GAP-43 expression due to cell death. Our previous studies have shown that by 3 weeks after injury, 90% of axotomized cortical transcallosal pyramidal cells within 1 mm of the wound have died w17x. This is consistent with the observations of others that closer proximity of axotomy can increase the chances that an intrinsic neuron will die
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w2,6x, as well as increasing the chances that it will respond regeneratively w10,37x. Thus, many of the axotomized cells scored here were undoubtedly destined to die. Overall, these results paint a picture of very little regenerative response by transcallosal cortical neurons to close axotomy. Some of this poor response may be speciesor strain-specific. For example, the mouse has been found to have lower expression levels of GAP-43 than the rat, both constitutively and in response to induction by kainate w28x. In addition, the mouse strain used in our experiments, C57 Blr6J, has been found to be the most ‘regeneration deficient’, with respect to sensory axon regeneration, of five different mouse strains examined w21,25,26x. However, there may be other reasons for the poor response, even to proximal axotomy, by these neurons. One possible explanation for the ‘proximity effect’ is that the presence of local axon collaterals may prevent regenerative responses. If axotomy provides a stimulus for growth due to a loss of axonally-provided factors, then the presence of remaining collaterals still attached to the cell body could override this loss. Pyramidal neurons in the mammalian cortex typically have extensive local axon collaterals quite close to the cell body w13,39x as well as in other layers of the cortex w23,33x. This morphology confers a richness of intrinsic circuits in the neocortex which probably contributes to its unique information processing capabilities w11x, and which may also contribute to the lack of regenerative response in cortical neurons, since axotomy that is even within several hundred microns of the soma still leaves an extensive network of axon collaterals attached to the soma. A recent report has shown that, in the rat, a different population of cortical projection neurons, corticospinals, did upregulate GAP-43 mRNA following very close axotomy achieved by devascularizing the cortex w14x. In these experiments, subcortical lesions that cut the corticospinal axons within a few hundred microns produced a mean grain density in axotomized corticospinal neurons that was 3- to 4-fold greater than the mean grain density of unaxotomized corticospinal neurons in control animals. The difference between these results and ours could be due to the difference in species used, a difference in average proximity of the axotomy, or a difference in the regenerative response of the particular subpopulation of cortical neurons. Another possibility is that effects of trauma other than axotomy Žsuch as inflammation or ischemia. might be involved in the observed difference between axotomized and control neurons. Elevation of GAP-43 mRNA can be induced by traumatic insults other than axotomy, such as local inflammatory responses w24x or kainate-induced excitotoxicity w7x. The lesions of Figuereido et al. w14x not only axotomized the cortical projection neurons, but also devascularized overlying cortex. Thus it is not clear whether the GAP-43 elevation in these experiments was due to axotomy alone, to ischemia, to inflammation associated with a local infarction, or to a combination of these
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factors. In our experiments, unaxotomized and axotomized neurons were equally close to the injury and its possible local effects. Comparison of axotomized and unaxotomized transcallosal neurons near the lesion with cortical neurons in the temporal region of the contralateral cortex, which were far away from the lesion, indicated that all three populations had similar grain densities and thus similarly low levels of GAP-43 expression. This indicates that proximity to the lesion alone did not elevate GAP-43. Other studies in rats have found significant sprouting by cortical neurons, either in response to peripheral nerve grafts w5x or following chronic injury w31x. In these studies, the neurons were only identified as pyramidal neurons in layer V w31x or layers II–V w5x, and were not identified as to which subpopulation they belonged or where they projected. Thus, the difference in the growth response seen by these investigators and the lack of response reported here, may be due to the difference in species, or to a difference in cortical subpopulation. Taken together with our previous studies of morphological changes after injury w16x, our present results are consistent with a description of transcallosal cortical neurons in mouse as extremely ‘regeneration deficient’. In the study of factors that contribute to the relatively poor regeneration of intrinsic neurons, a well described regeneration deficient model may prove very useful. Future experiments will test whether the introduction of exogenous trophic factors can induce this population of cells to increase their regenerative response to axotomy.
Acknowledgements We thank A. Padwarthan for help with reduction of the image analysis data and C. Matthews for careful reading of the manuscript. This work was supported by the VA Research Service and a VA Merit Award.
References w1x Aguayo, A.J., Axonal regeneration from injured neurons in the adult mammalian central nervous system. In C.W. Cotman ŽEd.., Synaptic Plasticity, Guilford, New York, 1985, pp. 457–484. w2x Aguayo, A.J., Rasminsky, M., Bray, G.M., Carbonetto, S., McKerracher, L., Villegas-Perez, M.P., Vidal-Sanz, M. and Carter, D.A., Degenerative and regenerative responses of injured neurons in the central nervous system of adult mammals, Phil. Trans. R. Soc. Lond., 331 Ž1991. 337–342. w3x Barron, K.D., Comparative observations on the cytologic reactions of central and peripheral nerve cells to axotomy. In C.C. Kao, R.P. Bunge and P.J. Reier ŽEds.., Spinal Cord Reconstruction, Raven Press, New York, 1983, pp. 7–40. w4x Barron, K.D., Neuronal responses to axotomy: consequences and possibilities for rescue from permanent atrophy or cell death, Neural Regeneration and Transplantation, Alan R. Liss, Inc., 1989, pp. 79–99. w5x Benfey, M. and Aguayo, A.J., Extensive elongation of axons from rat brain into peripheral nerve grafts, Nature, 296 Ž1982. 150–152.
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Research report
Effect of proximal axotomy on GAP-43 expression in cortical neurons in the mouse E.J. Elliott b
a,b,)
, D.A. Parks
a,b
, P.S. Fishman
a,b
a Research SerÕice, VA Medical Center, 10 N. Greene Street, Baltimore, MD 21201, USA Department of Neurology, UniÕersity of Maryland at Baltimore, Baltimore, MD 21201, USA
Accepted 30 December 1996
Abstract As an approach to understanding why central neurons fail to regenerate, we have studied the response to proximal axotomy of transcallosal neurons of the cerebral cortex of the mouse. Anatomical studies have indicated only very slight regenerative responses by this population of cortical neurons. To further examine the regenerative response of these cells, we have looked by in situ hybridization at the expression of GAP-43 mRNA following axotomy caused by a stab wound delivered within about 200 mm to 1.25 mm of the cell body. Axotomized transcallosal neurons were compared with near-by unaxotomized transcallosal neurons, as well as with distant unaxotomized cortical neurons in the contralateral hemisphere. All three populations of neurons had been pre-labeled with Fluoro-Gold to allow identification. No up-regulation of GAP-43 mRNA above background levels was detected for axotomized cortical neurons at 1, 3 or 7 days after injury. In contrast, increases in mean silver grain density of up to 8-fold were measured in axotomized spinal cord motor neurons used as positive controls. Thus, as a population, the transcallosal cortical pyramidal neurons did not show a significant regenerative response, as monitored by GAP-43 upregulation, even with very close axotomy. These results identify this population of neurons as among the least regenerative studied, and suggest that, on a molecular level, inherent neuronal properties play a role in the limited regenerative response to brain injury. Keywords: Axonal regeneration; GAP-43; Axotomy; Injury; CNS; Cortical neuron
1. Introduction Neurons completely within the central nervous system Žinstrinsic neurons. of adult mammals fail to regenerate severed axons, while neurons with axons in the periphery Žextrinsic neurons. generally sprout after axotomy and grow new axons w29x. Both local environmental factors and inherent neuronal characteristics contribute to this difference w4,6,12x. Changing the local environment of intrinsic neurons by grafting embryonic or peripheral nerve tissue into the CNS can result in sprouting and axonal growth by the intrinsic neurons into the graft w1,15x. However, neurons in different regions of the CNS show different capacities for growth into transplants w1,15,27x. For example, neurons in phylogenetically newer regions of the brain such as the neocortex have generally shown less regenerative response than those in other regions w4x. One measure of the inherent capacity for regeneration is
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Corresponding author. Fax: q1 Ž410. 605-7906.
the extent to which axotomized neurons respond by upregulating genes and proteins associated with axonal growth, in particular, the growth associated-protein GAP-43. Axotomy of extrinsic neurons is followed by several- to many-fold increases in levels of GAP-43 mRNA or protein w18,34x. Early experiments found that intrinsic neurons failed to upregulate GAP-43 after axotomy w20,30,34x, but more recently it has been demonstrated that a number of intrinsic neurons will both sprout and upregulate GAP-43 and other growth-associated proteins, albeit transiently in some cases, if the lesion occurs close enough to the cell body. Examples include retinal ganglion cells following proximal axotomy of the optic nerve w10x, rubrospinal motor neurons in the midbrain following cervical spinal lesions w35x, and thalamic neurons after a near-by thalamic stab wound w38x. In working toward the goal of understanding why intrinsic neurons fail to regenerate and discovering how they can be stimulated to regenerate, it can be instructive to study the best possible examples of regenerative failure, such as neurons of the cerebral cortex w4x. We have chosen
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 1 0 0 - 5
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to study transcallosal pyramidal neurons, a subpopulation of cortical neurons which project axons across the corpus callosum to the contralateral cerebral cortex. This population offers the opportunity for close axotomy Žby a stab wound. with relatively little disruption of the surrounding tissue, as well as the possibility for a control population of near-by, unaxotomized transcallosal neurons. This population also has many cellular features that are shared with cortical pyramidal neurons in higher species, including humans, where axotomy due to brain trauma or stroke has devastating clinical consequences. Anatomical studies of transcallosal cortical neurons have provided evidence for only a very slight regenerative response after axotomy in the hemisphere contralateral to the cell body, with only modest axonal sprouting amidst a preponderance of axonal degeneration w16x. We wished to further examine the regenerative response of these cells by looking at expression of GAP-43 after more proximal axotomy, especially in light of a recent report of upregulation of GAP-43 in another subpopulation of cortical pyramidal neurons, corticospinal neurons, following extremely proximal axotomy w14,36x.
2. Materials and methods 2.1. Animal surgery Adult male mice, C57rBl6 strain, 2–3 months old, from Charles River Labs, were used in all experiments. Mice were anesthetized by methoxyflurane inhalation before and during all surgeries. After surgery, mice received an intramuscular injection of the analgesic butorphanol tartrate, 0.1 ml of 0.1 mgrml. The general strategy was to label transcallosal pyramidal neurons so that they could be positively identified later in coronal sections of the brain, and then to deliver a knife wound that would axotomize some of these pre-labelled neurons, and leave others intact. Fig. 1 illustrates the labelling and injury procedures. Transcallosal pyramidal neurons in the right parietal cortex were labeled with the fluorescent dye Fluoro-Gold, which is taken up by axons and transported retrogradely to the cell body w32x. A gelfoam sponge saturated with 4% Fluoro-Gold in distilled water was placed, after a large Ž3 = 4 mm. craniectomy, on the surface of the left parietal cortex. The gelfoam was held in place by tacking it to the cranium with cyanoacrylate glue, and the overlying skin was sutured closed. This procedure resulted in homotopic labeling of a large number of transcallosal neurons in the contralateral cortex, as well as labeling of numerous cortical neurons in the ipsilateral cortex. One week after Fluoro-Gold labeling, a second surgery was performed in which the fiber tracts underlying the parietal cortex were completely transected by a 5 mm long knife wound through the right parietal cortex, 3 mm to the
Fig. 1. This line drawing of a coronal brain section illustrates the labeling of transcallosal cortical neurons with Fluoro-Gold ŽFG. applied to the contralateral surface of the brain in a gelfoam planchet, and the severing of axons of the most laterally located of those neurons with a knife wound.
right of the midline. This lesion typically was located within a large field of pre-labeled neurons. Neurons lateral to the lesion had their axons severed by it Ži.e. were axotomized., while neurons located medial to the lesion remained connected to their targets in the contralateral hemisphere, and served as unaxotomized controls. Although the axotomized pyramidal cell bodies were located not more than 100 to 500 mm from the site of the stab wound, the axotomy produced proximal axons which varied in length from about 200 mm to as much as 1.25 mm, depending on the exact location of the cell body in the cortex. This variability arises because the transcallosal axons travel ventrally down through the cortex to reach the subcortical fiber tracts and then turn toward the midline. Thus, more superficial cell bodies, and more laterally placed cell bodies, had longer segments of proximal axon remaining after the stab wound than did deeper or more medial cell bodies. 2.2. Histologic procedures Animals were sacrificed by anesthetic overdose at 1, 3 or 7 days after the knife wound. These time points were chosen after consideration of the time course of GAP-43 mRNA upregulation reported in other regenerating systems, in which maximal GAP-43 mRNA upregulation occurs from one day to one week after injury w8,10,22,35x. The chest was opened, a catheter inserted into the aorta via the left cardiac ventricle and the vascular system was perfused with 25 ml of cold 10% sucrose-PBS, followed by 25 ml of 10% formalin, 10% sucrose-PBS. Brains were removed and postfixed for 4 h at 48C in 10% formalin-30% sucrose-PBS. Postfixation was found to be necessary to stabilize the Fluoro-Gold sufficiently that it would be retained throughout the lengthy incubations and rinsings of the in situ hybridization ŽISH. procedure. Even so, there was considerable loss of fluorescence.
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Brains were frozen on dry ice immediately after the 4 h post-fix, or were immersed overnight in 30% sucrose-PBS, for additional cryoprotection, before being frozen on dry ice. Coronal sections Ž14 mm. were cut on a cryostat, and thaw-mounted onto Fisher Superfrost coated slides. Every fifth slide was examined to verify that the wound completely transected the fiber tracts and that there were Fluoro-Gold-labelled neuronal cell bodies both medial and lateral to the wound. The remaining slides were stored at y808C until use. For use as positive controls for sensitivity of detection of GAP-43 mRNA in the ISH experiments, motor neurons in the spinal cord were axotomized by sciatic nerve crush. At the time of the crush, 2 ml of a 4% solution of Fluoro-Gold was injected inside the perineurial sheath and proximal to the lesion, using a hand-held Hamilton syringe. One week later, animals were perfused and the lumbar spinal cord was removed, post-fixed, cryoprotected and sectioned. Sections containing Fluoro-Gold-labelled neuron cell bodies were used in ISH experiments. Unlabelled, unaxotomized motor neurons on the contralateral side of the same sections were made visible by counter staining with Cresyl violet and were recognizable by their large size and characteristic position. 2.3. In situ hybridization Frozen brain sections and spinal cord sections, sealed in boxes with silica gel dessicant, were thawed at room temperature. Thawed sections were treated with proteinase K Ž5 mgrml in PBS. at 378C for 4 min, acetylated with 0.25% acetic anhydride in 0.1 M TEA, dehydrated through graded ethanols, delipidated in chloroform, and allowed to air dry. Dried sections were circled with a PAP pen ŽResearch Products International Corp., Mt. Prospect, Illinois.. The resulting outline of hydrophobic residue around each section allowed the two sections on each slide to be hybridized to 50 ml droplets of two different solutions- one containing 33P-labelled GAP-43 probe and a second containing 1600-fold excess unlabelled probe in addition to the labelled probe. This provided a negative control on each slide for specificity of probe binding. Any signal that remained in the ‘excess-cold’ section was considered nonGAP-43-specific. All sections were incubated 1 h at room temperature in a prehybridization mixture Ž47% formamide, 10% dextran, 4 = SSC buffer w150 mM NaCl, 15 mM sodium citrate, pH 7.0x, 1 = Denhardt’s reagent, 0.25 mgrml yeast tRNA, 0.5 mgrml sheared and boiled herring sperm DNA, 10 mM Tris, 1 mM EDTA. which contained no probe but did contain poly dA Ž5 mgrml, Boehringer-Mannheim Corporation, Indianapolis, IN, USA.. The sections were then incubated overnight at 378C in humidified chambers in the above hybridization medium containing 5 = 10 7 cpmrml Ž0.5 pmolrml. of 33 P-labelled GAP-43 probe, or contain-
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ing 33 P-labelled probe plus 800 pmolrml of unlabelled GAP-43 probe. The GAP-43 antisense probe sequence was complementary to bases 210–248 of mouse GAP-43 mRNA w9x. The oligonucleotide was synthesized and purified by the Biopolymer Laboratory of the University of Maryland at Baltimore, and labelled at the 3X-end using terminal deoxynucleotidyl transferase ŽBoerhinger-Mannheim. and walpha- 33 PxdATP ŽNew England Nuclear, Boston, MA, USA. to a specific activity of 1–3 =10 9 c.p.m.r mg. After overnight incubation, the slides were rinsed in 1 = SSC at room temperature, four changes of 0.5 = SSC at 528C in a shaking water bath, and two changes of 0.5 = SSC at room temperature. Sections were dehydrated through ethanols, air dried and dipped in liquid emulsion ŽNTB2 nuclear track emulsion, Kodak, Rochester, NY, USA. diluted 1:1 with sterile water. Emulsion-coated slides were exposed for 4–11 days in the dark at 48C, developed in Kodak D-19 developer Ždiluted 1:1., dehydrated, cleared in xylene, and coverslipped with D.P.X. neutral mounting medium ŽAldrich Chemical Co., Milwaukee, WI, USA.. Sections were viewed both with epi-illumination through a UV filter set and with bright field illumination. Fluorescent images and bright field images were captured using a Cohu video camera, Scion frame grabber, and IP Lab Spectrum image analysis software by Signal Analytics ŽVienna, VA, USA.. Fields of view were chosen for image analysis which offered a high density of fluorescentlylabelled cells. Cells chosen for analysis were those with a relatively uniform size Ž8–12 mm long. and shape Žpyramidal to elliptical.. Silver grains over Fluoro-Gold-labelled cells were counted by superimposing fluorescent and bright field images. Cellular cross-sectional areas were computed from outlines that were hand-drawn around the fluorescent cell bodies. Within these cellular outlines, the area occupied by silver grains was measured by the computer and converted to a grain count by dividing the grains area by a mean grain size calculated by the software for that particular image. This grain count was expressed as an absolute number of grains per ‘cell profile’ Ži.e. grains per section of cell., and as a grain density. For each animal, grains over ; 30 axotomized cortical neurons and ; 30 control, non-axotomized neurons were counted on the same slide, and means were calculated and compared for significant difference using a two-tailed ttest. The ratio of the two means was determined for each slide, thus normalizing slide-to-slide variations in background signal. Eight to nine animals were examined at each time point. Background levels of silver grain density could not be quantitatively determined by measuring grain density from regions near the fluorescently labelled axotomized and non-axotomized neurons, because these regions might contain transcallosal cortical neurons which had not filled with Fluoro-Gold or which had lost the dye during the ISH protocol. Two different estimates of background grain
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density were obtained- one from Fluoro-Gold labelled neurons in the excess-cold sections, and a second from a population of Fluoro-Gold labelled neurons located in the temporal region of the contralateral cerebral cortex. This second population of neurons representated cortical pyramidal neurons distant from the wound. As a positive control for detection of GAP-43 mRNA, spinal cord sections containing axotomized motor neurons were included in the ISH experiments. It has been established that spinal motor neurons upregulate GAP-43 mRNA following sciatic nerve section w22x. The extent of upregulation in the spinal motor neuron controls was determined by comparing grain density over axotomized motor neurons with grain density over unaxotomized motor neurons on the contralateral side. The unaxotomized cell bodies, which were not fluorescently labeled, were visualized by light counterstaining with cresyl violet. In 3 out of 15 spinal motor neuron preparations that were examined,
GAP-43 upregulation, as well as faint Fluoro-Gold labelling, was observed in motor neurons contralateral to the axotomized motor neurons. Such a contralateral effect has been reported by others w22x and might be due to the presence of very fine collaterals that extend to the contralateral side. None of these animals were used as positive controls for ISH experiments.
3. Results After Fluoro-Gold application to the left parietal cortex, fluorescent transcallosal pyramidal neurons were found at depths corresponding to layers II, III and V of the right parietal cortex. Since sections were not stained to show cell bodies, it was not possible to assign cells unequivocally to a particular layer. However, these assignments agree with anatomical studies of rat callosal projections
Fig. 2. Light micrographs of coronal sections through the cerebral cortex of a mouse 1 day after a stab wound to the parietal region. Transcallosal neurons were pre-labelled with Fluoro-Gold 7 days before the wound. Unaxotomized cortical neurons, medial to the wound, are shown with epi-fluorescent illumination in ŽA., and the same field of view is shown with simultaneous brightfield and fluorescent in ŽB.. Axotomized cortical neurons, lateral to the wound, are shown in epi-fluorescent illumination in ŽC., and the same field of view is shown with simultaneous brightfield and fluorescent illumination in ŽD.. There is no obvious grain clustering over either unaxotomized or axotomized transcallosal neurons. Calibration bars s 20 mm.
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w19,40x. Fluoro-Gold-labelled neurons generally showed both diffuse, cytoplasmic-wide fluorescence and punctate vesicular fluorescence Žsee Fig. 2A.. Following a stab wound to the right parietal cortex, fluorescent pyramidal cells medial to the wound appeared intact and similar to labeled neurons in uninjured animals. Lateral to the wound, the number Žper unit area. of fluorescent cells was generally much less than that of cells medial to the wound. Moreover, the number of fluorescent lateral Žaxotomized. cells was generally smaller at 7 days following injury than at 1 day. This is consistent with our observation that many transcallosal neurons die following proximal axotomy. After in situ hybridization, there was no obvious grain clustering over axotomized transcallosal neurons, in any of the animals at any of the post-lesion times ŽFig. 2.. Thus it was clear that there was no upregulation of GAP-43 that approached the level seen in spinal cord motor neurons, for which grain clustering was obvious ŽFig. 3.. To look more closely for possible smaller changes in GAP-43 levels, mean silver grain counts were compared for axotomized and unaxotomized neurons in each of the 26 animals. The mean grain density for both axotomized and unaxotomized neurons was in the range of background grain density, as estimated from excess-cold sections and from cortical neurons distant to the wound. Thus, it was not possible to subtract this background value without arriving at negative values in many cases. When mean grain densities, uncorrected for background, were compared, axotomized cortical neurons did not differ significantly from unaxotomized cells in the majority of animals ŽFig. 4A.. This contrasted with the spinal cord control preparations, in which axotomized motor neurons showed from 2- to 8-fold increases in silver grain density compared to unaxotomized motor neurons. Seven of the 26 experimental animals Žindicated with
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asterisks in Fig. 4A. showed differences in grain density between axotomized and unaxotomized cortical neurons that were statistically significant Ž P - 0.0006.. However, these differences included both increases and decreases, and were modest in size compared with the positive controls. For example, for the cortical preparation with a 70% higher grain density in axotomized neurons, the matched spinal cord control gave an 8-fold higher grain density over axotomized motor neurons. Moreover, neither the magnitude nor direction of the differences correlated with probable length of proximal axon for the cells involved. Cell swelling is a well characterized part of the response of extrinsic neurons to axotomy w3x. If cell swelling occurred for the transcallosal cortical neurons in these experiments, then the amount of mRNA per unit volume Žor per unit cross-sectional area. would be diluted, and estimates of mRNA based on density would be underestimates. Fig. 4B shows a plot of the mean cross-sectional cellular area, expressed as the ratio of axotomized to unaxotomized cells, vs. time after injury, for the 26 animals studied. For most animals, the ratio was about 1.0, indicating no change in cell size. However, in 7 of the 26 animals, the difference in mean cross-sectional area between axotomized cells and unaxotomized cells was statistically significant Ž P - 0.003.. In all these cases, the mean axotomized cell area was 20 to 30% higher, suggesting cell swelling after axotomy. The animals with statistically significant differences are indicated with asterisks in Fig. 4B. Five of these seven were among the seven animals that showed changes Žeither increases or decreases. in silver grain density with axotomy. If cell swelling did occur, then actual grain counts per cell are a more accurate criterion for comparison than grain density. Fig. 4C shows the mean grain count per cell profile, for axotomized compared to unaxotomized cells. Grains per cell cross section are presented here, rather than
Fig. 3. Light micrographs of a cross-section of a spinal cord from a mouse 7 days after unilateral sciatic nerve crush. The section has been probed with the 33 P-labeled GAP-43 oligonucleotide probe and stained with cresyl violet. Axotomized spinal cord motor neurons Žin A. have an 8-fold greater density of silver grains than unaxotomized spinal cord motor neurons on the contralateral side Žin B.. Arrows point to examples of motor neuron cell bodies. Calibration bar is equal to 20 mm.
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grains per cell volume, because the nucleus was not visible in these unstained preparations and thus cytoplasmic volume could not be accurately estimated. The results based on grain countsrcell are similar to those based on grain density, that is, in most animals the grain counts are the same for axotomized and unaxotomized cells, though there are a few individuals with statistically significant differences, two with higher mean grain counts for axotomized cells and two with lower. The data from one of these individuals, with a 2-fold greater mean grain count for axotomized cells, is depicted in Fig. 5. This scatter plot of grain count vs. cellular cross-sectional area for individual Fig. 5. Scatter plot of grain count vs. cellular cross-sectional area for individual cortical neurons in an animal for which the mean grain count of axotomized neurons was 2-fold greater than that of unaxotomized neurons Žsame animal as illustrated in Fig. 2.. Time after axotomy s 7 days.
neurons shows that, in this animal, higher grain counts do not correlate with larger cross-sectional cellular area.
4. Discussion
Fig. 4. The ratio of the mean values for axotomized neurons compared to unaxotomized neurons, in each of the 26 animals studied, are plotted for: ŽA. silver grain density, ŽB. cellular cross-sectional area, and ŽC. silver grain count per cellular cross-scetion. Asterisks mark those animals for which the difference of the ratio from 1.0 was statistically significant Ž P - 0.0005 in A, P - 0.003 in B, P - 0.006 in C..
The GAP-43 measurements reported here extend previous morphological evidence that transcallosal neurons in the cerebral cortex show little regenerative response to axotomy w16x. That earlier work looked at transcallosal cells axotomized in the contralateral hemisphere, within 5 to 10 mm of the cell body. The work reported here looks at transcallosal cells axotomized in the ipsilateral hemisphere, within 200 mm to 1.25 mm of the cell body. These latest results show that, even with very proximal axotomy, there did not appear to be meaningful differences in GAP-43 mRNA expression between axotomized and unaxotomized transcallosal cortical neurons. In the few Ž4 out of 26. individuals for which there were statistically significant differences in mean grain counts for axotomized compared with unaxotomized neurons, the biological significance of the difference was not clear, since in all cases the grain counts were within the range of background levels, and in two cases the axotomized value was higher and in two cases it was lower. Of the two animals with higher axotomized grain counts, one was taken at 1 day after axotomy and the other at 7 days, and of the two animals with lower grain counts, one was at 1 day and the other at 7 days. Thus, there is no correlation with time after injury, and no ‘trend’ in the data, as might be seen if there were transient upregulation andror if there were a decline in GAP-43 expression due to cell death. Our previous studies have shown that by 3 weeks after injury, 90% of axotomized cortical transcallosal pyramidal cells within 1 mm of the wound have died w17x. This is consistent with the observations of others that closer proximity of axotomy can increase the chances that an intrinsic neuron will die
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w2,6x, as well as increasing the chances that it will respond regeneratively w10,37x. Thus, many of the axotomized cells scored here were undoubtedly destined to die. Overall, these results paint a picture of very little regenerative response by transcallosal cortical neurons to close axotomy. Some of this poor response may be speciesor strain-specific. For example, the mouse has been found to have lower expression levels of GAP-43 than the rat, both constitutively and in response to induction by kainate w28x. In addition, the mouse strain used in our experiments, C57 Blr6J, has been found to be the most ‘regeneration deficient’, with respect to sensory axon regeneration, of five different mouse strains examined w21,25,26x. However, there may be other reasons for the poor response, even to proximal axotomy, by these neurons. One possible explanation for the ‘proximity effect’ is that the presence of local axon collaterals may prevent regenerative responses. If axotomy provides a stimulus for growth due to a loss of axonally-provided factors, then the presence of remaining collaterals still attached to the cell body could override this loss. Pyramidal neurons in the mammalian cortex typically have extensive local axon collaterals quite close to the cell body w13,39x as well as in other layers of the cortex w23,33x. This morphology confers a richness of intrinsic circuits in the neocortex which probably contributes to its unique information processing capabilities w11x, and which may also contribute to the lack of regenerative response in cortical neurons, since axotomy that is even within several hundred microns of the soma still leaves an extensive network of axon collaterals attached to the soma. A recent report has shown that, in the rat, a different population of cortical projection neurons, corticospinals, did upregulate GAP-43 mRNA following very close axotomy achieved by devascularizing the cortex w14x. In these experiments, subcortical lesions that cut the corticospinal axons within a few hundred microns produced a mean grain density in axotomized corticospinal neurons that was 3- to 4-fold greater than the mean grain density of unaxotomized corticospinal neurons in control animals. The difference between these results and ours could be due to the difference in species used, a difference in average proximity of the axotomy, or a difference in the regenerative response of the particular subpopulation of cortical neurons. Another possibility is that effects of trauma other than axotomy Žsuch as inflammation or ischemia. might be involved in the observed difference between axotomized and control neurons. Elevation of GAP-43 mRNA can be induced by traumatic insults other than axotomy, such as local inflammatory responses w24x or kainate-induced excitotoxicity w7x. The lesions of Figuereido et al. w14x not only axotomized the cortical projection neurons, but also devascularized overlying cortex. Thus it is not clear whether the GAP-43 elevation in these experiments was due to axotomy alone, to ischemia, to inflammation associated with a local infarction, or to a combination of these
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factors. In our experiments, unaxotomized and axotomized neurons were equally close to the injury and its possible local effects. Comparison of axotomized and unaxotomized transcallosal neurons near the lesion with cortical neurons in the temporal region of the contralateral cortex, which were far away from the lesion, indicated that all three populations had similar grain densities and thus similarly low levels of GAP-43 expression. This indicates that proximity to the lesion alone did not elevate GAP-43. Other studies in rats have found significant sprouting by cortical neurons, either in response to peripheral nerve grafts w5x or following chronic injury w31x. In these studies, the neurons were only identified as pyramidal neurons in layer V w31x or layers II–V w5x, and were not identified as to which subpopulation they belonged or where they projected. Thus, the difference in the growth response seen by these investigators and the lack of response reported here, may be due to the difference in species, or to a difference in cortical subpopulation. Taken together with our previous studies of morphological changes after injury w16x, our present results are consistent with a description of transcallosal cortical neurons in mouse as extremely ‘regeneration deficient’. In the study of factors that contribute to the relatively poor regeneration of intrinsic neurons, a well described regeneration deficient model may prove very useful. Future experiments will test whether the introduction of exogenous trophic factors can induce this population of cells to increase their regenerative response to axotomy.
Acknowledgements We thank A. Padwarthan for help with reduction of the image analysis data and C. Matthews for careful reading of the manuscript. This work was supported by the VA Research Service and a VA Merit Award.
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