- Email: [email protected]
Initiation and time course of mitosis of non-neuronal cells after spinal motoneuron axotomy
Brain Research, 178 (1979) 519-528 © Elsevier/North-Holland Biomedical Press
519
I N I T I A T I O N A N D T I M E COURSE OF MITOSIS OF N O N - N E U R O N A L CELLS A F T E R SPINAL M O T O N E U R O N A X O T O M Y
DAVID L. McILWAIN and PAUL B. FAREL Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, N. C. 27514 (U.S.A.)
(Accepted April 5th, 1979) Key words:
neuronal regeneration - - motoneuron, frog - - axotomy - - microglia - [SH]thymidine
SUMMARY The mitotic response of non-neuronal cells following motor axon transection was measured after in vitro incorporation of [3H]thymidine in frog spinal cord. This predominantly ipsilateral response occurs more rapidly and is of greater magnitude when motor axons are unilaterally transected at the ventral root than after sciatic nerve transection. N o increase in incorporation occurred when regenerating fibers were transected a second time before reinnervation, but an increase was observed when the second operation was performed after the formation of functional neuromuscular connections had taken place. Autoradiographic studies after dorsal or ventral root transection showed that the distribution of labeled cells approximated the anatomical extent of the injured cellular elements within the spinal cord. These data are discussed in relation to the characteristics of the dividing cells and the nature of the events eliciting mitosis.
INTRODUCTION Motoneurons respond to transection of their axons with dramatic changes in their metabolism, structure and physiological properties (see reviews in refs. 9 and 12). At least some of these changes may be required for successful axonal regeneration. In addition, non-neuronal cells located near the injured neuron show increased mitotic activity. These non-neuronal cells have been reported to include microglial4,1s, ~2 and perhaps other cell types, such as capillary endothelia 17 and hematogenous cells1, 24 (cf. ref. 16), although doubt exists as to the exact identity of the dividing cells 11,1s. The relation of non-neuronal cell division to other aspects of the axon reaction is unclear. Watson 22 has emphasized the close temporal association between microglial cell proliferation and the loss of synaptic boutons on the surface of the regenerating neuron. This association may mean that microglia play a role in desynapsis z, although they may also serve other functions during the axon reactiong, 20.
520 The present study examines the initiation and time course of the mitotic response of non-neuronal cells following motoneuron axotomy by means of [:lH]thymidine incorporation detected by liquid scintillation counting or autoradiography. These experiments were undertaken to obtain data which might be related to studies in progress of electrophysiological and biochemical features of the axon reaction. Frog spinal cord was chosen for these studies both for the ease with which it can be maintained in vitro and because of the unique advantages it offers for electrophysiological analysis7, 8. The data obtained following ventral root transection are contrasted with those collected following dorsal root transection. METHODS Grass frogs (Rana pipiens) obtained from Carolina Biological (Burlington, N.C.) were housed in cages through which water at 22-26 °C was periodically flushed. Room temperature ranged from 18 to 26 °C. Animals were fed ground calves' liver 2-3 times each week. Details of the surgical procedures appear elsewhere8. The ninth ventral root (using the embryonic numbering system) was unilaterally transected just proximal to its exit from the vertebral canal. The cut ends of the roots separated 1-2 mm, providing assurance that the transection was complete. In other preparations, the dorsal root or sciatic nerve was transected instead of the ventral root. After sacrifice, the appropriate region of spinal cord was removed from the frog and freed of spinal roots in oxygenated frog Ringer solution buffered with 5 mM sodium phosphate and containing 1 ~ glucose (pH 7.3). This medium was routinely used in the experiments which follow. The segment was hemisected and one side was marked with India ink. In control experiments, India ink did not affect the incorporation of [3H]thymidine into the marked spinal tissue. Statistical comparisons between groups in these experiments involved application of two-tailed t-tests.
Radiometric analysis of spinal tissue After being separated and weighed, the operated and control sides of the spinal cord ( ~ = 5.7 mg for each hemisegment) were transferred together into 2 ml of fresh, chilled and oxygenated frog Ringer solution in a 10 ml beaker. The incorporation of [3H]thymidine in vitro was highly temperature dependent, with a Q10 of at least 12. For this reason, experimental and control tissue were always incubated simultaneously in the same vessel. Two #Ci of [3H]thymidine (New England Nuclear Corp.) were added to the medium, which was then shaken and capped with Parafilm. Samples from up to six frogs were incubated at 12 ± 4 °C for 19 4- 4 h on a Model SKI2 Stir Cool apparatus (Thermoelectrics Unlimited, Inc.). Oxygen was slowly bubbled through the solutions via thin Nalgene tubing. After incubation, the tissue was removed, washed twice with 2 ml of fresh Ringer solution, and each piece was transferred to a separate 6 ml disposable glass test tube to which 2 ml of frog Ringer was then added. The tissue was homogenized by sonication for 1-2 min in a Model 9 bath-type sonicator (Heat Systems-Ultrasonics, Inc.) under conditions previously shown to liberate nuclei from
521 spinal tissue 13. The homogenates were spun at 900 × g for 2 min, the supernatants discarded and the pellets washed twice with fresh Ringer solution, using the sonicator for resuspension of the pellet and centrifugation as above. The washed pellets were then dissolved in 0.25 ml of Protosol (New England Nuclear Corp.), which required at least 30 min with occasional vortex mixing, and transferred to counting vials with two 2.5 ml aliquots of scintillation cocktail (1.0~ PPO, 0.025~ dimethyl POPOP in toluene) and allowed to stabilize in a refrigerated Searle Mark II scintillation counter for 3 h before counting. The two wash steps for the homogenates were performed in all experiments reported here, although it has since been found that the same results are obtained after centrifugation of the unwashed homogenate in cold 0.2N HC 104 and direct analysis of the pellet. The latter procedure substantially increases the yield of sedimentable, labeled material and is also less time-consuming. In order to verify the incorporation of label into DNA, washed pellets were suspended in 0.5 ml of 0.43 M Tris.HC1 (pH 7.8) containing 5.4 mM MgCI2 and 4.3 mM CaC12. Bovine pancreatic DNAase II (100 /A; Sigma Chemical Co.), freshly dissolved in 0.15 M NaC1 (1300 U/ml) was added to the suspension and the mixture incubated 2 h at 38 °C. The suspension was chilled on ice for 30 min, spun for 2 min at 900 × g, and the pellet washed and prepared for scintillation counting as above. Autoradiography In most experiments in which spinal tissue was prepared for autoradiography, [3H]thymidine was incorporated by the in vitro procedure just described, except that the segments were not hemisected before incubation. After the labeled tissue had been washed twice with chilled frog Ringer solution, it was fixed for 2-3 days at 4 °C in 2 glutaraldehydeq3.1 m sodium phosphate buffer (pH 7.0), followed by 4-5 days at room temperature. It was then dehydrated and embedded in paraffin and crosssections (10 #m) were coated with Kodak NTE-2 photographic emulsion, using a modification of the procedure of Cowan et al. 4, Cresyl violet was used as a counterstain. Preparations showed numerous labeled cells after one week of exposure. There were no further qualitative changes following 3 weeks' additional exposure. In two frogs, spinal cord was labeled in vivo by injection of 1 #Ci of [~H]thymidine/g body weight into the ventral lymph sac one week after unilateral ventral root section. Four weeks postoperatively segment 9 was removed from each frog and the tissue prepared for autoradiography as above.
RESULTS Response to ventral root transection At varying intervals following unilateral transection of the ninth ventral root (motoneuron axotomy), the ninth spinal segment was removed, cut longitudinally along the midline and incubated with [SH]thymidine. The radioactivity incorporated by the two sides was determined as the ratio of cpm per mg wet tissue on the experimental to control sides (E/C ratio) and plotted as a function of post-operative time (Fig. 1). The E/C ratio was greatest at 5-8 days and declined to values typical of
522
6 O_
I
I
VENTRALROOT
4
tLl
I
I
i
I
I-2
3-4
5-6
7-8
I
I
I
9-10 11-13 14-16 DAYS POST-AXOTOMY
[
17-20
//
J
34
Fig. 1. Ipsilateral mitotic response to ventral root 9 transection. [aH]Thymidine incorporation is expressed as the ratio of cpm/mg wet weight of the operated to the unoperated side. Mean values :5 S.E.M. are given for groups of 9-14 frogs at each time point except day 34, where four frogs were studied. The shaded line represents 4- 1 S:E.M. around the mean ratios obtained for unoperated animals (left to right hemisegments). Each hemisegment contained an average of 240 total cpm over blanks.
unoperated animals by about day 35. The data varied considerably from animal to animal, even in single groups of frogs analyzed simultaneously; e.g. the E/C ratio at day 7 ranged from 1.25 to I0.1 ( ~ 4- S.E.M. ~- 5.3 4- 0.83). Variation in E/C ratios obtained among cords incubated on the same day could not be attributed to differences in spinal wet weight, in incubation conditions, or in length of the proximal stump. This variability may account for failure of the scintillation counting method to detect the small increases in the incorporation of label seen by autoradiography on the side opposite the transection vs unoperated controls. Autoradiograms were prepared from frog lumbar enlargements 5-6 days following ventral root transection and after 19-20 h incubation in vitro with [3H]thymidine. By this time, the motoneurons of the operated side consistently had a swollen appearance, enlarged nucleoli and moderate chromatolysis, as also found by Price and Porter 15. Previous work s showed no change in motoneuron numbers following axotomy when appropriate corrections for changes in cell size were made. Autoradiographic grains were predominant on the operated side and were located primarily in the gray matter over small cells near the motoneurons (Fig. 2a). This localization of label was also seen in frog spinal cord labeled in vivo one week postoperatively and examined at the end of the fourth week and is similar to the localization reported for axotomized mammalian cranial motoneurons ls,~°. That the label was incorporated primarily into D N A is supported by the fact that DNAase treatment of homogenized, labeled spinal cords from two frogs sacrificed 5 days after ventral root transection resulted in an average E/C ratio by scintillation counting of 1.5, compared to an E/C ratio of 5.3 for untreated preparations from similar animals. Estimates of label distribution based upon observations of 1180 labeled cells (Fig. 2b) from 2 frogs showed that 73 ~ of all grain clusters was found in the ventral quadrant of the spinal cord on the side of the operation. Nineteen percent of the total labeled cells was in the dorsal ipsilateral quadrant. The remainder of labeled cells
523
b
Fig. 2. a: autoradiograph of motoneuron pool 6 days after ventral root transection. Arrows indicate labeled cells. The swollen motoneurons contain eccentric nuclei with enlarged nucleoli. Note the accumulation of Nissl substance in the periphery of the topmost motoneuron. The bar represents 20 #m. b: distribution of 1180 labeled cells 6 days after transection of the left ventral root. At this time, most of the labeled cells in the ipsilateral ventral quadrant were not immediately adjacent to motoneuron somata. (about 8 ~ ) was equally distributed between the two contralateral quadrants. Thus, there were over 11 times as many labeled cells on the operated as on the unoperated side, compared with an E/C ratio of about 5 on the sixth post-operative day when cpm/mg was the unit of reference. Because the dendritic processes of motoneurons of the ninth segment are confined to the ipsilateral side (ref. 19 and Farel, unpublished H R P observations), the role played by mitotic cells on the contralateral side of the cord is unclear. Similar contralateral effects have been noted following axotomy of rat hypoglossal motoneurons 20 and spinal motoneurons 1°. Approximately 11 ~ of the labeled cells in the dorsal and ventral quadrants of the operated side was clearly adjacent to blood vessels, while only 3-4 ~ was similarly situated on control side. [3H]Thymidine is also incorporated into non-neuronal cells which proliferate during neuronal degeneration (Fig. 3). When dorsal roots of the ninth segment in three frogs were severed, separating primary afferent axons and synapses from their cell bodies, E/C ratios of [3H]thymidine incorporation 5-6 days later averaged about 3.5. However, autoradiograms showed labeled cells after dorsal root transection to be highly localized to the ipsilateral dorsal quadrant of the ninth segment, reflecting the location of degenerating axons and terminals. Ninety-four per cent of the grain
524
b
Fig. 3. a: autoradiograph of dorsal horn ipsilateral to dorsal root transection performed 6 days previously. Arrows indicate labeled cells. The central canal is visible in the lower right hand corner. The bar represents 20/zm. b: distribution of labeled cells 6 days after transection of the left dorsal root. Of the 724 labeled cells counted, one cell was in the contralateral dorsal quadrant and four were in the contralateral ventral quadrant.
clusters was in the ipsilateral dorsal quadrant and 5~o in the ipsilateral ventral quadrant (Fig. 3b). Once again the E/C ratios from the scintillation method were much lower than those calculated from autoradiographic data. This discrepancy may have resulted from the superior detection of background radioactivity by the scintillation method which, if the same on the two sides, would lower the E/C ratio.
Response to sciatic nerve transection The mitotic response was influenced by the distance between the axonal transection site and the affected cell body. Fig. 4 shows the time course of [3H]thymidine incorporation into ipsilateral spinal segments 9 and 10 after unilateral transection of the sciatic nerve at mid thigh. When these data are compared to those in Fig. 1, the latency is seen to increase about 3-fold while the magnitude of the mitotic response is approximately halved. The peak response now appears at the 13th to 14th post-operative day, and [ZH]thymidine incorporation then returns to normal by the 35th post-operative day. Reinjury before functional reinnervation Regenerating spinal motor axons in the frog can traverse the site of ventral root
525
4 0
~2 bJ
.,/j I 5-6
I 7-8
I 9-10
I I 11-12 15-14 DAYS
I I 15-17 18-21 POST-AXOTOMY
I 22-25
/ ~
I 26-5[
Fig. 4. Ipsilateral mitotic response to mid thigh sciatic nerve transection. The data are expressed as in Fig. 1, with mean values 4- S.E.M. for groups of 3-11 frogs (£,1 = 6) at each time point. The shaded line represents the mean values 4- S.E.M. for unoperated animals.
transection and at least some of them eventually reinnervate skeletal muscle after about 50 days s. In an attempt to reinitiate the mitotic response before and after functional reinnervation had occurred, frogs with unilateral ventral root lesions were subjected to a second transection 30 days after the first operation, while others were reoperated between 75 and 230 days later. Presence ofreinnervation was tested by stimulation of the peripheral stump of the ninth spinal nerve. All animals were then tested for [aH]thymidine incorporation 6 days after the second operation. In five animals whose ventral roots were recut on day 30, i.e. after the mitotic response to the first operation had largely subsided (Fig. l) but before functional reinnervation had occurred, no statistically significant secondary increase in [aH]thymidine incorporation was observed (E/C = 1.24 4- 0.11 ; 0.2 > P > 0.1). On the other hand, when the interval between operations was 75 days or more, an increase in mitotic activity was noted on the reoperated side (E/C = 2.73 4- 0.45; P < 0.01). The response was not as large as that observed after the first operation nor did it occur as regularly (range: 0.63-5.26). Moreover, the response did not increase between day 75 and day 230 after the first operation. A similar variation in recovery of ventral root reflex activity has been observed in electrophysiological experimentsS, 9. DISCUSSION The experiments presented here provide new information about the time course and anatomical distribution of mitosis of non-neuronal cells following ventral root transection in frog. In addition, the effects on mitosis of distance of the lesion from the motoneuron somata and of a second transection of the m o t o r fibers are quantified. These data will be discussed in relation to the characteristics of the dividing cells and in relation to the nature of the event which initiates mitosis. The tendency of labeled cells to follow the pattern of dendritic arborization of frog spinal motoneurons found at the ninth segment 19 suggests that cell division is facilitated by proximity to some part of the injured neuron. A comparison, then, of the latencies and peaks of changes within the motoneuron with those of mitosis might be expected to shed light on the motoneuronal activities necessary to elicit cell division.
526 However, such comparisons of motoneuronal changes with non-neuronal mitosis encompass a variety of techniques, not all of which necessarily have the same sensitivity. Finding that the latency of a motoneuronal change is different from that of mitosis is important only to the extent one can be sure that this difference is not due to unequal sensitivities of the techniques employed. A further difficulty arises when comparisons of duration and time to peak of motoneuronal and mitotic responses are attempted. Many motoneuronal responses are measured cumulatively while [3H]thymidine incorporation provides a measure of mitotic rate over the incubation period. Nevertheless, it is of interest to note that, among the earliest responses of frog motoneurons to axotomy is increased acid phosphatase activity 20 hours after ventral root transection 3. RNA content and cell volume increase within 4 days of a sciatic nerve crush, peak at 30-40 days, and return to normal by 151 days ~, as do other morphological responses 1~. A more fruitful methodological approach to the question of the relation of motoneural changes to the mitotic response is to attempt a dissociation by experimental manipulation. For example, Watson 2° found that increasing the distance between the somata of rat hypoglossal neurons and the site of transection altered the latency of mitosis, but not that of desynapsis. Similarly, resectioning previously severed hypoglossal fibers before reconnection to muscle produces a resurgence of RNA synthesis 21, but as in the present experiments, no increase in non-neuronal mitotic activity 23. The latencies to onset of mitosis provide a basis for estimating the m i n i m u m rate of travel of the signal for cell division. The minimum rates of travel of the signal (calculated by dividing response latency by distance of the injury from the cell body) were 2.3 ram/day for ventral root transections (7 mm from the cell body) and 7.4 ram/day for sciatic nerve transections (67 mm from the cell body) based on response latencies of 3 and 9 days, respectively. Several possible mechanisms may contribute to the disparity between these estimates. For example, the signal may travel along the ventral root and peripheral nerve portions of the motor axons at different rates. Further, the existence of a delay between the time the signal reaches the cell body and the onset of mitosis would result in an underestimation of the speed of the signal in both experiments, and, depending upon the characteristics of the delay, may affect the two estimates in an unequal manner. In spite of these uncertainties, the calculated speeds correspond to the 4-5 mm/day Cragg ~ calculated on the basis of Watson's 21 data for speed of the signal for increased nucleolar RNA synthesis following crush of rat hypoglossal nerve. Similar calculations based on Watson's 20 data for non-neuronal cell division suggest a signal speed of 4-7 ram/day. Both scintillation counting and autoradiography demonstrate the presence of mitotic cells following either dorsal root or ventral root transection, despite the fact that these two operations have markedly different consequences for the injured neurons. Dorsal root transection produces degeneration of intraspinal processes while few if any motoneurons degenerate following ventral root transection s. Labeled cells are thus found around both degenerating primary afferent processes and regenerating motoneurons. These data raise questions concerning the function of the mitotic cells,
527 particularly whether they play a role that is unique to regenerative activities of injured neurons. Watson z0 found labeled cells surrounding axotomized motoneurons regardless of whether a particular neuron was going to regenerate its axon or die and be phagocytosed. Dividing cells have been suggested to participate in the loss of synapses around axotomized motoneurons 2, to act as macrophages 20, and to help meet the metabolic demands of the regenerating neuronsg, 20. However, the role of mitotic cells will not become clear until it is ascertained whether the same cell type is involved in all the situations in which cell division has been reported and whether a single cell type can serve more than one function. ACKNOWLEDGEMENTS
We are grateful to Ms Sibyl Bemelmans for assistance in the operation and care of animals used in this study and to Ms Carol Metz for aid with histological procedures. Supported by NSF Grant BNS76-24528 and USPHS Grant NS12103. Ancillary support was provided by USPHS Grants NS11132 and NS14899. REFERENCES 1 Adrian, E. K., Jr. and Smothermon, R. D., Leucocytic infiltration into the hypoglossal nucleus following injury to the hypoglossal nerve, Anat. Rec., 166 (1974) 99-116. 2 Blinzinger, K. and Kreutzberg, G., Displacement of synaptic terminals from regenerating motoneurons by microglial cells, Z. Zellforsch., 85 (1968) 145-157. 3 Cerf, J. A. and Chacko, L. W., Retrograde reaction in motoneuron dendrites following ventral root section in the frog, J. comp. Neurol., 109 (1958) 205-219. 4 Cowan, W. M., Gottlieb, D. I., Hendrickson, A. E. and Woolsey, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1973) 21-51. 5 Cragg, B. G., What is the signal for chromatolysis? Brain Research, 23 (1970) 1-21. 6 Edstr6m, J. E., Riboneucleic acid changes in the motoneurons of the frog during axon regeneration, J. Neurochem., 5 (1959) 43-49. 7 Farel, P. B., Selectivity in the loss of synaptic input to frog spinal motoneurons following ventral root section, Neurosci. Abstr., 4 (1978) 530. 8 Farel, P. B., Reflex activity of regenerating frog spinal motoneurons, Brain Research, 158 (1978) 331-341. 9 Grafstein, B., The nerve cell body response to axotomy, Exp. Neurol., 48 (1975) 32-51. 10 Kerns, J. M. and Hinsrnan, E. J., Neuroglial response to sciatic neurectomy I. Light microscopy and autoradiography, J. comp. Neurol., 151 (1974) 237-254. 11 Kerns, J. M. and Hinsman, E. J., Neuroglial response to sciatic neurectomy II. Electron microscopy, J. comp. Neurol., 151 (1974) 255-280. 12 Lieberman, A. R., The axon I eaction: a review of the principal features of perikaryal response to injury, Int. Rev. Neurobiol., 14 (1971) 49-124. 13 Mcllwain, D. L. and. Capias-Covey, P., The nuclear D N A content of large ventral spinal neurons, J. Neurochem., 27 (1976) 109-112. 14 Price, D. L., The response of amphibian glial cells to axonal transection, J. Neuropath. exp. Neurol., 31 (1972) 267-277. 15 Price, D. L. and Porter, K. R., The response of ventral horn neurons to axonal transection, J. Cell Biol., 53 (1972) 24-37. 16 Schelper, R. L., Adrian, E. K., Jr. and Wiliams, M. G., A comparison of the types and distribution of labeled cells found in injured nervous tissue using aI-I-thymidine or 125I-deoxyuridine before injury, Ant. Rec., 187 (1977) 707. 17 Sj6strand, J., Studies on glial cells in the hypoglossal nucleus of the rabbit during nerve regeneration and morphological changes in glial cells during nerve regeneration, Acta physiol, scand., 67, Suppl. 270 (1966) 1-43.
528 18 Sumner, B. E. H., The nature of the dividing cells around axotomized hypoglossal neurones, J. Neuropath. exp. NeuroL, 33 (1974) 507-518. 19 Szekely, G., The morphology of motoneurons and dorsal root fibers in the frog's spinal cord, Brain Research, 103 (1976) 275-290. 20 Watson, W. E., An autoradiographic study of the incorporation of nucleic acid precursors by neurones and glia during nerve regeneration, J. PhysioL (Lond.), 180 (1965) 741-753. 21 Watson, W. E., Observations on the nucleolar and total cell body nucleic acid of injured nerve cells, ./. PhysioL (Lond.), 196 (1968) 655-676. 22 Watson, W. E., Cellular responses to axotomy and to related procedures, Brit. Med. Bull., 30 (1974) 112-115. 23 Watson, W. E., Cell Biology of Brain, John Wiley and Sons, Inc., New York, 1976, p. 252. 24 Young, M. B., H3T-Labelled blood cells in the CNS response to axotomies at various times after isotope injection, J. Neuropath. exp. NeuroL, 36 (1977) 465-473.
519
I N I T I A T I O N A N D T I M E COURSE OF MITOSIS OF N O N - N E U R O N A L CELLS A F T E R SPINAL M O T O N E U R O N A X O T O M Y
DAVID L. McILWAIN and PAUL B. FAREL Department of Physiology, University of North Carolina School of Medicine, Chapel Hill, N. C. 27514 (U.S.A.)
(Accepted April 5th, 1979) Key words:
neuronal regeneration - - motoneuron, frog - - axotomy - - microglia - [SH]thymidine
SUMMARY The mitotic response of non-neuronal cells following motor axon transection was measured after in vitro incorporation of [3H]thymidine in frog spinal cord. This predominantly ipsilateral response occurs more rapidly and is of greater magnitude when motor axons are unilaterally transected at the ventral root than after sciatic nerve transection. N o increase in incorporation occurred when regenerating fibers were transected a second time before reinnervation, but an increase was observed when the second operation was performed after the formation of functional neuromuscular connections had taken place. Autoradiographic studies after dorsal or ventral root transection showed that the distribution of labeled cells approximated the anatomical extent of the injured cellular elements within the spinal cord. These data are discussed in relation to the characteristics of the dividing cells and the nature of the events eliciting mitosis.
INTRODUCTION Motoneurons respond to transection of their axons with dramatic changes in their metabolism, structure and physiological properties (see reviews in refs. 9 and 12). At least some of these changes may be required for successful axonal regeneration. In addition, non-neuronal cells located near the injured neuron show increased mitotic activity. These non-neuronal cells have been reported to include microglial4,1s, ~2 and perhaps other cell types, such as capillary endothelia 17 and hematogenous cells1, 24 (cf. ref. 16), although doubt exists as to the exact identity of the dividing cells 11,1s. The relation of non-neuronal cell division to other aspects of the axon reaction is unclear. Watson 22 has emphasized the close temporal association between microglial cell proliferation and the loss of synaptic boutons on the surface of the regenerating neuron. This association may mean that microglia play a role in desynapsis z, although they may also serve other functions during the axon reactiong, 20.
520 The present study examines the initiation and time course of the mitotic response of non-neuronal cells following motoneuron axotomy by means of [:lH]thymidine incorporation detected by liquid scintillation counting or autoradiography. These experiments were undertaken to obtain data which might be related to studies in progress of electrophysiological and biochemical features of the axon reaction. Frog spinal cord was chosen for these studies both for the ease with which it can be maintained in vitro and because of the unique advantages it offers for electrophysiological analysis7, 8. The data obtained following ventral root transection are contrasted with those collected following dorsal root transection. METHODS Grass frogs (Rana pipiens) obtained from Carolina Biological (Burlington, N.C.) were housed in cages through which water at 22-26 °C was periodically flushed. Room temperature ranged from 18 to 26 °C. Animals were fed ground calves' liver 2-3 times each week. Details of the surgical procedures appear elsewhere8. The ninth ventral root (using the embryonic numbering system) was unilaterally transected just proximal to its exit from the vertebral canal. The cut ends of the roots separated 1-2 mm, providing assurance that the transection was complete. In other preparations, the dorsal root or sciatic nerve was transected instead of the ventral root. After sacrifice, the appropriate region of spinal cord was removed from the frog and freed of spinal roots in oxygenated frog Ringer solution buffered with 5 mM sodium phosphate and containing 1 ~ glucose (pH 7.3). This medium was routinely used in the experiments which follow. The segment was hemisected and one side was marked with India ink. In control experiments, India ink did not affect the incorporation of [3H]thymidine into the marked spinal tissue. Statistical comparisons between groups in these experiments involved application of two-tailed t-tests.
Radiometric analysis of spinal tissue After being separated and weighed, the operated and control sides of the spinal cord ( ~ = 5.7 mg for each hemisegment) were transferred together into 2 ml of fresh, chilled and oxygenated frog Ringer solution in a 10 ml beaker. The incorporation of [3H]thymidine in vitro was highly temperature dependent, with a Q10 of at least 12. For this reason, experimental and control tissue were always incubated simultaneously in the same vessel. Two #Ci of [3H]thymidine (New England Nuclear Corp.) were added to the medium, which was then shaken and capped with Parafilm. Samples from up to six frogs were incubated at 12 ± 4 °C for 19 4- 4 h on a Model SKI2 Stir Cool apparatus (Thermoelectrics Unlimited, Inc.). Oxygen was slowly bubbled through the solutions via thin Nalgene tubing. After incubation, the tissue was removed, washed twice with 2 ml of fresh Ringer solution, and each piece was transferred to a separate 6 ml disposable glass test tube to which 2 ml of frog Ringer was then added. The tissue was homogenized by sonication for 1-2 min in a Model 9 bath-type sonicator (Heat Systems-Ultrasonics, Inc.) under conditions previously shown to liberate nuclei from
521 spinal tissue 13. The homogenates were spun at 900 × g for 2 min, the supernatants discarded and the pellets washed twice with fresh Ringer solution, using the sonicator for resuspension of the pellet and centrifugation as above. The washed pellets were then dissolved in 0.25 ml of Protosol (New England Nuclear Corp.), which required at least 30 min with occasional vortex mixing, and transferred to counting vials with two 2.5 ml aliquots of scintillation cocktail (1.0~ PPO, 0.025~ dimethyl POPOP in toluene) and allowed to stabilize in a refrigerated Searle Mark II scintillation counter for 3 h before counting. The two wash steps for the homogenates were performed in all experiments reported here, although it has since been found that the same results are obtained after centrifugation of the unwashed homogenate in cold 0.2N HC 104 and direct analysis of the pellet. The latter procedure substantially increases the yield of sedimentable, labeled material and is also less time-consuming. In order to verify the incorporation of label into DNA, washed pellets were suspended in 0.5 ml of 0.43 M Tris.HC1 (pH 7.8) containing 5.4 mM MgCI2 and 4.3 mM CaC12. Bovine pancreatic DNAase II (100 /A; Sigma Chemical Co.), freshly dissolved in 0.15 M NaC1 (1300 U/ml) was added to the suspension and the mixture incubated 2 h at 38 °C. The suspension was chilled on ice for 30 min, spun for 2 min at 900 × g, and the pellet washed and prepared for scintillation counting as above. Autoradiography In most experiments in which spinal tissue was prepared for autoradiography, [3H]thymidine was incorporated by the in vitro procedure just described, except that the segments were not hemisected before incubation. After the labeled tissue had been washed twice with chilled frog Ringer solution, it was fixed for 2-3 days at 4 °C in 2 glutaraldehydeq3.1 m sodium phosphate buffer (pH 7.0), followed by 4-5 days at room temperature. It was then dehydrated and embedded in paraffin and crosssections (10 #m) were coated with Kodak NTE-2 photographic emulsion, using a modification of the procedure of Cowan et al. 4, Cresyl violet was used as a counterstain. Preparations showed numerous labeled cells after one week of exposure. There were no further qualitative changes following 3 weeks' additional exposure. In two frogs, spinal cord was labeled in vivo by injection of 1 #Ci of [~H]thymidine/g body weight into the ventral lymph sac one week after unilateral ventral root section. Four weeks postoperatively segment 9 was removed from each frog and the tissue prepared for autoradiography as above.
RESULTS Response to ventral root transection At varying intervals following unilateral transection of the ninth ventral root (motoneuron axotomy), the ninth spinal segment was removed, cut longitudinally along the midline and incubated with [SH]thymidine. The radioactivity incorporated by the two sides was determined as the ratio of cpm per mg wet tissue on the experimental to control sides (E/C ratio) and plotted as a function of post-operative time (Fig. 1). The E/C ratio was greatest at 5-8 days and declined to values typical of
522
6 O_
I
I
VENTRALROOT
4
tLl
I
I
i
I
I-2
3-4
5-6
7-8
I
I
I
9-10 11-13 14-16 DAYS POST-AXOTOMY
[
17-20
//
J
34
Fig. 1. Ipsilateral mitotic response to ventral root 9 transection. [aH]Thymidine incorporation is expressed as the ratio of cpm/mg wet weight of the operated to the unoperated side. Mean values :5 S.E.M. are given for groups of 9-14 frogs at each time point except day 34, where four frogs were studied. The shaded line represents 4- 1 S:E.M. around the mean ratios obtained for unoperated animals (left to right hemisegments). Each hemisegment contained an average of 240 total cpm over blanks.
unoperated animals by about day 35. The data varied considerably from animal to animal, even in single groups of frogs analyzed simultaneously; e.g. the E/C ratio at day 7 ranged from 1.25 to I0.1 ( ~ 4- S.E.M. ~- 5.3 4- 0.83). Variation in E/C ratios obtained among cords incubated on the same day could not be attributed to differences in spinal wet weight, in incubation conditions, or in length of the proximal stump. This variability may account for failure of the scintillation counting method to detect the small increases in the incorporation of label seen by autoradiography on the side opposite the transection vs unoperated controls. Autoradiograms were prepared from frog lumbar enlargements 5-6 days following ventral root transection and after 19-20 h incubation in vitro with [3H]thymidine. By this time, the motoneurons of the operated side consistently had a swollen appearance, enlarged nucleoli and moderate chromatolysis, as also found by Price and Porter 15. Previous work s showed no change in motoneuron numbers following axotomy when appropriate corrections for changes in cell size were made. Autoradiographic grains were predominant on the operated side and were located primarily in the gray matter over small cells near the motoneurons (Fig. 2a). This localization of label was also seen in frog spinal cord labeled in vivo one week postoperatively and examined at the end of the fourth week and is similar to the localization reported for axotomized mammalian cranial motoneurons ls,~°. That the label was incorporated primarily into D N A is supported by the fact that DNAase treatment of homogenized, labeled spinal cords from two frogs sacrificed 5 days after ventral root transection resulted in an average E/C ratio by scintillation counting of 1.5, compared to an E/C ratio of 5.3 for untreated preparations from similar animals. Estimates of label distribution based upon observations of 1180 labeled cells (Fig. 2b) from 2 frogs showed that 73 ~ of all grain clusters was found in the ventral quadrant of the spinal cord on the side of the operation. Nineteen percent of the total labeled cells was in the dorsal ipsilateral quadrant. The remainder of labeled cells
523
b
Fig. 2. a: autoradiograph of motoneuron pool 6 days after ventral root transection. Arrows indicate labeled cells. The swollen motoneurons contain eccentric nuclei with enlarged nucleoli. Note the accumulation of Nissl substance in the periphery of the topmost motoneuron. The bar represents 20 #m. b: distribution of 1180 labeled cells 6 days after transection of the left ventral root. At this time, most of the labeled cells in the ipsilateral ventral quadrant were not immediately adjacent to motoneuron somata. (about 8 ~ ) was equally distributed between the two contralateral quadrants. Thus, there were over 11 times as many labeled cells on the operated as on the unoperated side, compared with an E/C ratio of about 5 on the sixth post-operative day when cpm/mg was the unit of reference. Because the dendritic processes of motoneurons of the ninth segment are confined to the ipsilateral side (ref. 19 and Farel, unpublished H R P observations), the role played by mitotic cells on the contralateral side of the cord is unclear. Similar contralateral effects have been noted following axotomy of rat hypoglossal motoneurons 20 and spinal motoneurons 1°. Approximately 11 ~ of the labeled cells in the dorsal and ventral quadrants of the operated side was clearly adjacent to blood vessels, while only 3-4 ~ was similarly situated on control side. [3H]Thymidine is also incorporated into non-neuronal cells which proliferate during neuronal degeneration (Fig. 3). When dorsal roots of the ninth segment in three frogs were severed, separating primary afferent axons and synapses from their cell bodies, E/C ratios of [3H]thymidine incorporation 5-6 days later averaged about 3.5. However, autoradiograms showed labeled cells after dorsal root transection to be highly localized to the ipsilateral dorsal quadrant of the ninth segment, reflecting the location of degenerating axons and terminals. Ninety-four per cent of the grain
524
b
Fig. 3. a: autoradiograph of dorsal horn ipsilateral to dorsal root transection performed 6 days previously. Arrows indicate labeled cells. The central canal is visible in the lower right hand corner. The bar represents 20/zm. b: distribution of labeled cells 6 days after transection of the left dorsal root. Of the 724 labeled cells counted, one cell was in the contralateral dorsal quadrant and four were in the contralateral ventral quadrant.
clusters was in the ipsilateral dorsal quadrant and 5~o in the ipsilateral ventral quadrant (Fig. 3b). Once again the E/C ratios from the scintillation method were much lower than those calculated from autoradiographic data. This discrepancy may have resulted from the superior detection of background radioactivity by the scintillation method which, if the same on the two sides, would lower the E/C ratio.
Response to sciatic nerve transection The mitotic response was influenced by the distance between the axonal transection site and the affected cell body. Fig. 4 shows the time course of [3H]thymidine incorporation into ipsilateral spinal segments 9 and 10 after unilateral transection of the sciatic nerve at mid thigh. When these data are compared to those in Fig. 1, the latency is seen to increase about 3-fold while the magnitude of the mitotic response is approximately halved. The peak response now appears at the 13th to 14th post-operative day, and [ZH]thymidine incorporation then returns to normal by the 35th post-operative day. Reinjury before functional reinnervation Regenerating spinal motor axons in the frog can traverse the site of ventral root
525
4 0
~2 bJ
.,/j I 5-6
I 7-8
I 9-10
I I 11-12 15-14 DAYS
I I 15-17 18-21 POST-AXOTOMY
I 22-25
/ ~
I 26-5[
Fig. 4. Ipsilateral mitotic response to mid thigh sciatic nerve transection. The data are expressed as in Fig. 1, with mean values 4- S.E.M. for groups of 3-11 frogs (£,1 = 6) at each time point. The shaded line represents the mean values 4- S.E.M. for unoperated animals.
transection and at least some of them eventually reinnervate skeletal muscle after about 50 days s. In an attempt to reinitiate the mitotic response before and after functional reinnervation had occurred, frogs with unilateral ventral root lesions were subjected to a second transection 30 days after the first operation, while others were reoperated between 75 and 230 days later. Presence ofreinnervation was tested by stimulation of the peripheral stump of the ninth spinal nerve. All animals were then tested for [aH]thymidine incorporation 6 days after the second operation. In five animals whose ventral roots were recut on day 30, i.e. after the mitotic response to the first operation had largely subsided (Fig. l) but before functional reinnervation had occurred, no statistically significant secondary increase in [aH]thymidine incorporation was observed (E/C = 1.24 4- 0.11 ; 0.2 > P > 0.1). On the other hand, when the interval between operations was 75 days or more, an increase in mitotic activity was noted on the reoperated side (E/C = 2.73 4- 0.45; P < 0.01). The response was not as large as that observed after the first operation nor did it occur as regularly (range: 0.63-5.26). Moreover, the response did not increase between day 75 and day 230 after the first operation. A similar variation in recovery of ventral root reflex activity has been observed in electrophysiological experimentsS, 9. DISCUSSION The experiments presented here provide new information about the time course and anatomical distribution of mitosis of non-neuronal cells following ventral root transection in frog. In addition, the effects on mitosis of distance of the lesion from the motoneuron somata and of a second transection of the m o t o r fibers are quantified. These data will be discussed in relation to the characteristics of the dividing cells and in relation to the nature of the event which initiates mitosis. The tendency of labeled cells to follow the pattern of dendritic arborization of frog spinal motoneurons found at the ninth segment 19 suggests that cell division is facilitated by proximity to some part of the injured neuron. A comparison, then, of the latencies and peaks of changes within the motoneuron with those of mitosis might be expected to shed light on the motoneuronal activities necessary to elicit cell division.
526 However, such comparisons of motoneuronal changes with non-neuronal mitosis encompass a variety of techniques, not all of which necessarily have the same sensitivity. Finding that the latency of a motoneuronal change is different from that of mitosis is important only to the extent one can be sure that this difference is not due to unequal sensitivities of the techniques employed. A further difficulty arises when comparisons of duration and time to peak of motoneuronal and mitotic responses are attempted. Many motoneuronal responses are measured cumulatively while [3H]thymidine incorporation provides a measure of mitotic rate over the incubation period. Nevertheless, it is of interest to note that, among the earliest responses of frog motoneurons to axotomy is increased acid phosphatase activity 20 hours after ventral root transection 3. RNA content and cell volume increase within 4 days of a sciatic nerve crush, peak at 30-40 days, and return to normal by 151 days ~, as do other morphological responses 1~. A more fruitful methodological approach to the question of the relation of motoneural changes to the mitotic response is to attempt a dissociation by experimental manipulation. For example, Watson 2° found that increasing the distance between the somata of rat hypoglossal neurons and the site of transection altered the latency of mitosis, but not that of desynapsis. Similarly, resectioning previously severed hypoglossal fibers before reconnection to muscle produces a resurgence of RNA synthesis 21, but as in the present experiments, no increase in non-neuronal mitotic activity 23. The latencies to onset of mitosis provide a basis for estimating the m i n i m u m rate of travel of the signal for cell division. The minimum rates of travel of the signal (calculated by dividing response latency by distance of the injury from the cell body) were 2.3 ram/day for ventral root transections (7 mm from the cell body) and 7.4 ram/day for sciatic nerve transections (67 mm from the cell body) based on response latencies of 3 and 9 days, respectively. Several possible mechanisms may contribute to the disparity between these estimates. For example, the signal may travel along the ventral root and peripheral nerve portions of the motor axons at different rates. Further, the existence of a delay between the time the signal reaches the cell body and the onset of mitosis would result in an underestimation of the speed of the signal in both experiments, and, depending upon the characteristics of the delay, may affect the two estimates in an unequal manner. In spite of these uncertainties, the calculated speeds correspond to the 4-5 mm/day Cragg ~ calculated on the basis of Watson's 21 data for speed of the signal for increased nucleolar RNA synthesis following crush of rat hypoglossal nerve. Similar calculations based on Watson's 20 data for non-neuronal cell division suggest a signal speed of 4-7 ram/day. Both scintillation counting and autoradiography demonstrate the presence of mitotic cells following either dorsal root or ventral root transection, despite the fact that these two operations have markedly different consequences for the injured neurons. Dorsal root transection produces degeneration of intraspinal processes while few if any motoneurons degenerate following ventral root transection s. Labeled cells are thus found around both degenerating primary afferent processes and regenerating motoneurons. These data raise questions concerning the function of the mitotic cells,
527 particularly whether they play a role that is unique to regenerative activities of injured neurons. Watson z0 found labeled cells surrounding axotomized motoneurons regardless of whether a particular neuron was going to regenerate its axon or die and be phagocytosed. Dividing cells have been suggested to participate in the loss of synapses around axotomized motoneurons 2, to act as macrophages 20, and to help meet the metabolic demands of the regenerating neuronsg, 20. However, the role of mitotic cells will not become clear until it is ascertained whether the same cell type is involved in all the situations in which cell division has been reported and whether a single cell type can serve more than one function. ACKNOWLEDGEMENTS
We are grateful to Ms Sibyl Bemelmans for assistance in the operation and care of animals used in this study and to Ms Carol Metz for aid with histological procedures. Supported by NSF Grant BNS76-24528 and USPHS Grant NS12103. Ancillary support was provided by USPHS Grants NS11132 and NS14899. REFERENCES 1 Adrian, E. K., Jr. and Smothermon, R. D., Leucocytic infiltration into the hypoglossal nucleus following injury to the hypoglossal nerve, Anat. Rec., 166 (1974) 99-116. 2 Blinzinger, K. and Kreutzberg, G., Displacement of synaptic terminals from regenerating motoneurons by microglial cells, Z. Zellforsch., 85 (1968) 145-157. 3 Cerf, J. A. and Chacko, L. W., Retrograde reaction in motoneuron dendrites following ventral root section in the frog, J. comp. Neurol., 109 (1958) 205-219. 4 Cowan, W. M., Gottlieb, D. I., Hendrickson, A. E. and Woolsey, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1973) 21-51. 5 Cragg, B. G., What is the signal for chromatolysis? Brain Research, 23 (1970) 1-21. 6 Edstr6m, J. E., Riboneucleic acid changes in the motoneurons of the frog during axon regeneration, J. Neurochem., 5 (1959) 43-49. 7 Farel, P. B., Selectivity in the loss of synaptic input to frog spinal motoneurons following ventral root section, Neurosci. Abstr., 4 (1978) 530. 8 Farel, P. B., Reflex activity of regenerating frog spinal motoneurons, Brain Research, 158 (1978) 331-341. 9 Grafstein, B., The nerve cell body response to axotomy, Exp. Neurol., 48 (1975) 32-51. 10 Kerns, J. M. and Hinsrnan, E. J., Neuroglial response to sciatic neurectomy I. Light microscopy and autoradiography, J. comp. Neurol., 151 (1974) 237-254. 11 Kerns, J. M. and Hinsman, E. J., Neuroglial response to sciatic neurectomy II. Electron microscopy, J. comp. Neurol., 151 (1974) 255-280. 12 Lieberman, A. R., The axon I eaction: a review of the principal features of perikaryal response to injury, Int. Rev. Neurobiol., 14 (1971) 49-124. 13 Mcllwain, D. L. and. Capias-Covey, P., The nuclear D N A content of large ventral spinal neurons, J. Neurochem., 27 (1976) 109-112. 14 Price, D. L., The response of amphibian glial cells to axonal transection, J. Neuropath. exp. Neurol., 31 (1972) 267-277. 15 Price, D. L. and Porter, K. R., The response of ventral horn neurons to axonal transection, J. Cell Biol., 53 (1972) 24-37. 16 Schelper, R. L., Adrian, E. K., Jr. and Wiliams, M. G., A comparison of the types and distribution of labeled cells found in injured nervous tissue using aI-I-thymidine or 125I-deoxyuridine before injury, Ant. Rec., 187 (1977) 707. 17 Sj6strand, J., Studies on glial cells in the hypoglossal nucleus of the rabbit during nerve regeneration and morphological changes in glial cells during nerve regeneration, Acta physiol, scand., 67, Suppl. 270 (1966) 1-43.
528 18 Sumner, B. E. H., The nature of the dividing cells around axotomized hypoglossal neurones, J. Neuropath. exp. NeuroL, 33 (1974) 507-518. 19 Szekely, G., The morphology of motoneurons and dorsal root fibers in the frog's spinal cord, Brain Research, 103 (1976) 275-290. 20 Watson, W. E., An autoradiographic study of the incorporation of nucleic acid precursors by neurones and glia during nerve regeneration, J. PhysioL (Lond.), 180 (1965) 741-753. 21 Watson, W. E., Observations on the nucleolar and total cell body nucleic acid of injured nerve cells, ./. PhysioL (Lond.), 196 (1968) 655-676. 22 Watson, W. E., Cellular responses to axotomy and to related procedures, Brit. Med. Bull., 30 (1974) 112-115. 23 Watson, W. E., Cell Biology of Brain, John Wiley and Sons, Inc., New York, 1976, p. 252. 24 Young, M. B., H3T-Labelled blood cells in the CNS response to axotomies at various times after isotope injection, J. Neuropath. exp. NeuroL, 36 (1977) 465-473.