The Response of Rubrospinal Neurons to Axotomy in the Adult Opossum, Didelphis virginiana XIAO MING Xv AND GEORGEF. MARTIN Department
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METHODS Ten adult (female) opossums were anesthetized by sodium pentobarbitol(40 mg/kg) so that the T-10 or T-11 segment of the thoracic spinal cord could be exposed by laminectomy using sterile techniques. After stabilization in a stereotaxic frame, lo-20 ~1of 5% fast blue (FB) was injected into the spinal cord using a lo-p1 Hamilton syringe. In half the cases,the injections were made bilaterally, whereas in the remaining ones they were made unilaterally. In all cases, the surgical exposure was closed and the animals were returned to the vivarium under the care of a veterinarian. Seven days later they were reanesthetized for sterile surgery so that the rubrospinal tract could be cut unilaterally, four segments rostral to the injection(s). In the caseswith unilateral injections, the lesion was made ipsilateral to the injection. Between 30 (N = 9) and 60 (N = 1) days later, the animals were given an overdose of the anesthetic and perfused transcardially with saline followed by a 0.1 it4 citrate buffer-lo% formaldehyde solution. The brain and spinal cord were dissected out and immersed in the same buffer with 30% sucrose for approximately 24 h at 4°C. The brains were scored by a shallow cut on the side of the injection so that the laterality of the tissue sections could be determined after mounting. Frozen sections of the injected segment of the spinal cord, the lesion site, and the brain stem were cut in the coronal plane at 40 pm. The sections were mounted immediately and coverslipped with Entellan (Merck) for viewing and photography with a Leitz (Orthoplan) fluorescence microscope using the A cube of the Ploem illumination system. In
INTRODUCTION It has been reported that many rubrospinal neurons die after axotomy although the degree of cell death depends on the age of the animal and the distance from the cell body the axon is cut (5, 6, 11, 12). In fact, death of rubrospinal neurons after axotomy has been suggested to be a contributing factor in the failure of rubrospinal axons to regenerate in neonatal and adult rats (5, 6,12). As an extension of ongoing studies on rubrospinal plasticity in developing opossums (9,15), we have undertaken experiments designed to determine the degree to which rubrospinal neurons survive transection of their axons in the adult animal. As in other species, most rubrospinal axons course in the lateral funiculus contralatera1to their origin and terminate primarily within laminae V through VII (3). Few, if any, recross at spinal levels. Opossums are useful for studies of rubrospinal plasticity because the development of their rubrospinal tract occurs postnatally rather than prenatally as in placental mammals (3). Our strategy was to label rubrospinal neurons by injecting the long-lasting fluorescent marker fast blue
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(FB) (14) into the thoracic cord and, 7 days later, to lesion the rubrospinal tract four segments rostra1 to the injection. After 30-60 days, the animals were sacrificed so that the red nucleus on the side opposite the lesion could be searched for labeled neurons which were presumed to have survived the lesion. Our results suggest that most rubrospinal neurons survive axotomy in adult opossums and that cell death is not a major factor in the failure of the rubrospinal tract to regenerate (15).
To provide endpoints for developmental studies of rubrospinal plasticity in the North American opossum, we have attempted to determine the degree to which rubrospinal neurons survive axotomy in the adult animal. Bilateral or unilateral injections of the long-lasting fluorescent marker fast blue were made into the T-10 or the T- 11 segment of the spinal cord to label rubrospinal neurons, and 7 days later, the rubrospinal tract was cut unilaterally four segments rostra1 to the injection(s). In cases with unilateral injections, the lesion was made ipsilateral to the injection. The animals were allowed to survive for 30-60 days before being sacrificed and perfused so that sections through the red nuclei could be examined for labeled neurons. The results show that most axotomized neurons survived the lesion, suggesting that lesion-dependent cell death is not a major factor in the failure of the rubrospinal tract to regenerate in the adult animal. o 1990 Academic PMS, IW.
0014-4886/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form
FIG. 1. Fluorescence photomicrograph (excitation wavelength equals 360 nm) of the injections in the case plotted in Fig. 2 (A) and a darkfield photomicrograph of a section through the lesion in the same case (B). The position of the rubrospinal tract is outlined in A (arrow), whereas the central canal (CC) and ventromedian fissure (arrow) are indicated in B. The bar indicates 1 mm.
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Caudal FIG. 2. Plot of labeled rubrospinal (Ipsi.) to the lesion of the rubrospinal drawn in the squares.
neurons (dots) from every 10th section through the red nucleus contralateral (Contral.) and ipsilateral tract shown in Pig. 1B. Rostra1 and caudal are indicated and labeled neurons from selected areas are
600 500 m 5 400 73 & 300 e 2 200 100
FIG. 4. Histogram showing the mean number nal neurons contralateral (Contral.) and ipsilateral in all of the cases with bilateral injections. The standard deviation.
of labeled rubrospi(Ipsi.) to the lesion error bars indicate
all cases, the positions of rubral neurons labeled by FB contralateral to the lesion were recorded from every fifth section using an X-Y plotter attached to the microscope stage by position transducers. All labeled neurons were recorded regardless of their labeling intensity, including those not sectioned through the nucleus. If neurons were labeled ipsilateral to the lesion they were also plotted and in the cases with bilateral injections, labeled cells were counted on both sides from the plots. To determine if the size of rubrospinal neurons changed after axotomy, labeled neurons from selected medial and lateral areas of the red nucleus were measured on both sides from every fifth section in the cases with bilateral injections. This was accomplished by first tracing the outline of labeled neurons using a drawing tube attached to the fluorescence microscope so that their area could be measured with the aid of an interactive computer-assisted image analysis system (Magiscan, Nikon/Joyce Loebl). The drawings of labeled neurons were fed into a black and white television camera (Dage-MtI series 68, Newvicon), then through a realtime video processor (Nippon Avionics, Model Image Sigma) and finally into a computer (Magiscan 2A, JoyceLoebl) for digitization and analysis. The outline of each labeled cell was filled in by the computer which was in-
structed to separate dark areas (labeled cells) from light areas (non-cells). Calibration was done so that the area of the labeled cells was measured in square micrometers. Statistical analysis was accomplished using the “result” program supplied by Joyce-Loebl. The output from the computer gave the mean area of the labeled cells on each side and a histogram which showed their size and frequency distributions. As a preliminary measure of total neuronal population, the cell numbers in the red nucleus contralateral and ipsilateral to the lesion were estimated according to the following protocol. After the labeled neurons were plotted, photographed, and measured, the coverslips were removed and the sections were stained with cresyl violet so that neurons in the red nucleus could be counted. The same sections used for the counts of labeled neurons were used for the counts of Nissl-stained cells. Rubral neurons with clear nucleoli were recorded using the X-Y plotter referred to previously and then counted. Cytological criteria were used to distinguish neurons from neuroglia, so it is not likely that we failed to count neurons because of shrinkage. A paired Student t test was used to determine whether the difference in the number of rubral neurons on the two sides was statistically significant.
RESULTS In all cases subjected to bilateral injections of FB into the thoracic cord and unilateral lesions which included the rubrospinal tract, neurons were labeled bilaterally in the red nucleus. A fluorescence photomicrograph of the bilateral injections from one case is provided in Fig. 1A. Although considerable necrosis was produced by the injections, rubral labeling appeared comparable to that seen after injections that did not produce extensive damage. Figure 1B is a dark-field photomicrograph of the lesion and it can be seen that the dorsal part of the lateral funiculus, including the rubrospinal tract, was completely cut. There was no spread of FB to the lesion site. Although labeled neurons were found in generally comparable areas on the two sides, they appeared to be fewer in number and less intensely fluorescent on the side contralateral to the lesion (Figs. 2 and 3). The decrease in number was particularly obvious in the area outlined by the square in Fig. 2 and indicated by the arrow in Fig. 31. It should be noted that rubral neurons which project to
FIG. 3. Fluorescence photomicrographs of labeled rubrospinal neurons contralateral (left column) and ipsilateral (right column) to the lesion in the case plotted in Fig. 2 (excitation wavelength equals 360 nm). The sections shown here are adjacent to those plotted in Fig. 2. The same sections were not used for plotting and photography because of fading. The photomicrographs in the two columns are at approximately the same level. It was difficult to match them exactly because the red nucleus on the side contralateral to the lesion shrinks by about 10%. Rostra1 (Rost.) and caudal (Caud.) are indicated. The bar in section A indicates 200 wrn and can be used for all figures.
FIG. 5. Fluorescence photomicrographs (excitation wavelength eql lals 360 and ipsilateral (right column) to a lesion of the rubrospinal tract in a ca se with right and left in A are shown at higher power in C and E; the areas out1 ined by The bars in A and C indicate 200 Km; the bar in A can be used for B and that in
caudal segments of the spinal cord in the opossum are not as restricted in location as their counterparts in the placental mammals (7). The histogram in Fig. 4 shows the mean number of neurons labeled contralateral (Con-
nm) of labeled rubrospinal neurons contralateral (left column) bilateral injections. The areas outlined by the rectangles on the the rectangles on the left and right in B are shown in D and F. C can be used for D-F.
tral., x = 353) and ipsilateral (Ipsi., x = 469) to the lesion in all of the cases with bilateral injections and it can be concluded that most (approximately 75%) rubrospinal neurons survived the lesion. The Student t test
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h 300 f z' 200 100
FIG. 7. Histogram showing the mean number of rubrospinal neurons contralateral (Central.) and ipsilateral (Ipsi.) to the lesion in the cases used for fluorescent-labeled cell counts. Neurons with clear nuclei and nucleoli were counted after the coverslips had been removed and the sections stained with cresyl violet. The error bars indicate standard deviation.
showed that the difference between the two groups was statistically significant (P < 0.01). The neurons labeled contralateral to the lesion also appeared smaller than those labeled ipsilaterally (Fig. 5). That impression was verified by measuring labeled neurons from the medial and lateral areas of the red nucleus indicated by the rectangles in Fig. 5. The mean area of labeled neurons in the contralateral red nucleus from all of the cases with bilateral injections was 480 pm’, whereas that on the ipsilateral side was 645 pm2 (Fig. 6). The difference between the two groups was statistically significant (P < 0.01).
photomicrographs FIG. 8. Fluorescence era1 (B) to a lesion of the rubrospinal tract
Since it might be argued that the smaller number of labeled neurons contralateral to the lesion reflects transport failure rather than cell death, we counted the number of rubral neurons whose nucleoli could be identified in the same sections used to measure labeled neurons. To accomplish this, the coverslips were removed and the sections were stained with cresyl violet. On the side contralateral to the lesion, the mean number of stained neurons in all cases was 172, whereas on the ipsilateral side it was 210 (Fig. 7). The paired t test showed that the difference between the two groups was statistically significant (P < 0.01). In all cases subjected to unilateral injections and lesions, labeled neurons were present in the contralateral red nucleus and they appeared comparable in position, number, and size to those labeled contralateral to the lesion in the cases with bilateral injections. In one animal, FB spread to the side opposite the injection and rubra1 neurons were labeled on both sides (Fig. 8). As in the cases with bilateral injections, the neurons labeled contralateral to the lesion (Fig. 8A) were fewer in number, less intensely labeled, and smaller than those labeled ipsilaterally (Fig. 8B). This was particularly true in the area indicated by the arrow in Fig. 8A which corresponds to that similarly indicated in Fig. 31. In the animal allowed to survive for 60 days, the labeling was comparable to that seen after 30 days. This case was not included in the quantitative analysis. DISCUSSION Fast blue (FB) worked well in our experiments, but after long-term survivals the possibility of transneuronal labeling must be considered. Such labeling was not a major factor, however, since the number of neurons labeled in the normal red nucleus after bilateral injec-
(excitation wavelength equals 360 nm) of rubrospinal neurons labeled contralateral by an injection which spread to both sides. The bar in A indicates 200 pm.
(A) and ipsilat-
tions appeared comparable to that seen after shorter survival times. It has been reported that ipsilateral as well as contralateral rubrospinal neurons respond to lesions of the rubrospinal tract (1). If so, we may have underestimated the degree to which such neurons died or shrunk on the contralateral side. We did not limit our counts and measurements of fluorescent neurons to those sectioned through the nuclei or nucleoli because it is difficult to record the necessary detail without losing fluorescent labeling. It is recognized, however, that cell size may affect the probability of encountering neuronal profiles in section samples. Since labeled neurons contralateral to the lesion were smaller than those on the ipsilateral side, it is possible that we underestimated the number of surviving neurons. It appears that most rubrospinal neurons survive axotomy at caudal thoracic levels for at least 2 months and that lesion-induced cell death is not the most likely explanation for lack of rubrospinal regeneration during the same time period (15). Much of the previous work (5,6), emphasized that rubrospinal neurons die after axotomy at comparable levels in rats, but more recently it has been claimed that all of them survive for at least 20 weeks (10). The earlier results in the rat were based on lack of retrograde labeling with HRP and orthograde labeling with [3H]leucine, but it is possible that neurons which survive axotomy do not transport or metabolize those markers normally. Such a conclusion is supported by the fact that the studies claiming survival of all neurons used the fluorescent marker Fluoro-Gold in a prelabeling paradigm similar to ours (10). Our finding that many, if not all, axotomized rubrospinal neurons are smaller than normal has also been reported for the rat (1,4,5) and it has been shown in that species that RNA decreases (1,2). Although most rubrospinal neurons survived axotomy in our experiments, some apparently did not. That conclusion is based on differences in the number of labeled rubrospinal neurons contralateral and ipsilateral to the lesion in cases with bilateral injections of FB and ipsilatera1 lesions of the rubrospinal tract and on counts of rubra1 neurons in the same cases after the coverslips had been removed and the sections stained with cresyl violet. It should be noted that the counts made from the cresyl violet-stained sections underestimated the proportion of neurons that survived axotomy, since only neurons with clear nuclei and nucleoli were counted and only a limited number of them project to caudal levels of the cord. Although our counting methods do not provide absolute numbers, they do provide reasonable estimates. In any case, our results appear to differ from those referred to above (1,10) which suggest that all rubrospinal neurons survive axotomy for at least 20 weeks in the rat. In the latter experiments Fluoro-Gold was used to prelabel ru-
brospinal neurons and the animals were subjected to complete transection of the cord. More rubrospinal neurons die after axotomy in developing opossums (15) than in adult animals (present study) and, paradoxically, death of rubrospinal neurons is most extensive during the critical period for rubrospinal plasticity. We have interpreted these results to suggest that rubrospinal plasticity in the developing opossum is due primarily to growth of new axons around the lesion, not to regeneration of cut axons (15). It would be interesting to know why some rubrospinal neurons survive axotomy, whereas others do not. In contrast to the results reported here, very few ganglion cells survive transection of the optic nerve in adult rats and mice (13). Since retinal axons have few collaterals proximal to the lesion, death of axotomized ganglion cells might be due to a lack of sustaining collaterals. In contrast, rubrospinal neurons have collaterals in the brain stem (8) and some of the ones which innervate lumbar levels of the cord have collaterals at cervical levels (7). It is possible, therefore, that the neurons which survived in our experiments were those which had the greatest number of collaterals rostra1 to the lesion. Following this line of reasoning, it may be that neurons in the areas of greatest cell death had the fewest collaterals rostra1 to the lesion. The number of collaterals may correlate with access to some as yet unknown trophic factor. ACKNOWLEDGMENTS The authors thank Ms. Mary Ann Jarrell for expert technical assistance and for typing the manuscript, Mr. Karl Rubin for photographic help, Mr. Michael Pindzola for helping with the computer counts and measurements, and Dr. Michael Beattie for reading early versions of the manuscript. This investigation was supported by USPHS Grant NS-25095.
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