J. Anat. (1989), 164, pp. 201-213 201 With 16 figures Printed in Great Britain Neuroglial response to neuron injury. A study using intraneural injection of ricinus communis agglutinin-60
E. A. LING, C. Y. WEN*, J. Y. SHIEH*, T. Y. YICK AND S. K. LEONG Department of Anatomy, Faculty of Medicine, National University of Singapore, Singapore 0511 and * Department of Anatomy, College of Medicine, National Taiwan University, Taipei, Taiwan 10018 (Accepted 27 September 1988)
INTRODUCTION Several studies have shown that ricinus communis agglutinin (RCA), when injected into a nerve in minute amounts, is retrogradely transported by axons in the nerve, resulting in a selective destruction of the parental cell bodies. Thus, the administration of RCA into the vagus nerve would cause a selective destruction of the efferent neurons in the dorsal motor nucleus (Wiley, Blessing & Reis, 1982; Ling & Leong, 1987, 1988). Such a 'suicide transport' of the toxic lectin is also evident in sensory neurons (Yamamoto, Iwasaki & Konno, 1983, 1984; Johnson, Westrum, Henry & Canfield, 1985; Wiley & Oeltmann, 1986). Recently, the use of RCA has become increasingly important as a research tool for tracing the central projections of primary afferents of peripheral nerves (Yamamoto et al. 1983; Leong & Tan, 1987; Ling & Leong, 1987). While much is known about the consequent death of neurons following RCA application, little is known about the response of the non-neuronal cells either closely associated with, or in the vicinity of, the degenerating neurons. According to Yamamoto et al. (1984), the selective destruction of neurons in the trigeminal and dorsal root ganglia by RCA could possibly stimulate the capsule cells involved in the phagocytosis of the degenerating nerve cells. How the neuroglial cells in the central nervous system react to the presence of degenerating neurons induced by RCA application is unclear. In their light microscopic observation, Wiley et al. (1982) reported that "a glial reaction was evident" in the dorsal motor nucleus four to seven days after the injection of RCA into the vagus nerve in rats. However, the functional role of the glial cells in such a chemically induced neuronal degeneration is uncertain. A further study was made by Streit & Kreutzberg (1988), who described the rapid proliferation of, and phagocytosis by, microglia in the rat facial nucleus following RCA injection into the facial nerve. There was, however, no evidence of infiltration of mononuclear cells into the site of neuronal degeneration as noted by Ling & Leong (1987). The aim of the present study was to examine in a sequential manner the degenerative changes of the neurons poisoned by RCA, and to clarify further the neuroglial reaction, especially the involvement of microglial cells in such a degenerative process. The ventral horn neurons in the lumbosacral enlargement were used as a model for this study because they have been shown at light microscopic level to be selectively destroyed by RCA injection into the sciatic nerve (Wiley & Oeltmann, 1986). 202 E. A. LING AND OTHERS
MATERIALS AND METHODS Male Wistar rats ranging in weight between 200 and 250 g were used in this study. Under chloral hydrate anaesthesia, the right sciatic nerve was exposed after the separation of the gluteus maximus muscle. A total volume of 3 ,al of 0 05 % ricinus communis agglutinin-60 (RCA-60) (Lot No. P83X02, Seikagaku Kogyo Co Ltd, Japan) in 0 01 M phosphate buffer was injected into the nerve (2 jul into its tibial and 1 jul into its common peroneal component) with a Hamilton syringe. The injected site was dried with a cotton bud. Following the administration of RCA-60, each animal was given an intravenous injection of0 6 ml 20 % lactose, a procedure which was found to be quite effective in reducing the mortality rate of the animals (Ling & Leong, 1988). The animals were allowed to survive for 1, 3, 7, 15, 30 and 60 days before they were re-anaesthetised with chloral hydrate and perfused. At least three animals were used at each of the time intervals. In each case, the animal was perfused with 100 ml of Ringer's solution, followed by a mixed aldehyde solution, made up of 2 % paraformaldehyde and 3 % glutaraldehyde in 0-1 M cacodylate buffer adjusted to pH 7 2-7 4. In addition to the experimental animals, two normal animals, not injected with RCA-60, were also perfused to serve as controls. After perfusion, which lasted 30 minutes, the spinal cord extending from the lower lumbar to the upper sacral region was removed. Vibratome sections of 200,um thickness were prepared from the lumbosacral enlargement, and postfixed in 1 % osmium tetroxide in 0 1 M cacodylate buffer for 1 hour. After dehydration in a graded series of alcohol, the sections were embedded in Araldite mixture. Ultrathin sections were double stained with uranyl acetate and lead citrate and were examined and photographed with a JEOL 1200EX electron microscope. In addition to the above, 2 rats, each surviving 3, 5 and 7 days after the RCA-60 injection, were perfused with 10% neutral formalin. The spinal cord was removed and processed for light microscopy. Seven micrometer thick sections were prepared and stained with haematoxylin and eosin or with cresyl fast violet. To investigate the particular cell type predominantly involved in the glial reaction, Araldite-embedded sections of 1 am thickness were prepared and stained with methylene blue. All the various glial cell types and neurons in the ventral horn region outlined in Figure 1 were identified according to the criteria and method described earlier (Ling et al. 1973) and counted.
OBSERVATIONS Light microscopy As reported by Wiley & Oeltmann (1986), the present study showed that injection of RCA into the sciatic nerve caused the degeneration of ventral horn neuronal cells, primarily of the larger category. The earliest sign of neuronal degeneration in the ventral horn seen with the light microscope was observed in animals given RCA-60 injection and perfused three days later. This became more severe in the five days postoperative rats (Fig. 1). The neurons in the contralateral ventral horn appeared normal. At a higher magnification, the majority of the RCA-poisoned neurons were swollen with pale cytoplasm (Fig. 2). Occasional degenerating neurons, however, were dark and appeared to have shrunk (Fig. 2). None of the small neurons seemed to be affected. Enumeration of the various glial cell types in the ventral horn of three normal uninjected rats showed that the proportion of oligodendrocytes, astrocytes and Neuroglial response to neuron injury 203
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Fig 1 e ~ 3 > ' ,~ : k -j Fig. 1. Transverse section of the lower lumbar cord segment 5 days after RCA injection showing selective neuronal destruction (arrows) in the ventral horn ipsilateral to RCA injection into the sciatic nerve. The neurons on the contralateral side are not affected (asterisks). The area outlined is the region in which all the various glial cell types are quantified in semithin sections. CC, central canal. Fig. 2. Higher magnification of the ventral horn of lower lumbar spinal cord 5 days after RCA injection. Arrows indicate some swollen degenerating neurons. A darkened degenerating neuron is indicated by an asterisk. Note that the smaller neurons are unaffected in the upper part of the picture. Glial reaction is evident in the circled area near the degenerating neurons. Fig. 3. Axon terminals (A) containing round and flattened synaptic vesicles are presynaptic to the soma (s) of a normal neuron. x 17500. 204 E. A. LING AND OTHERS Table 1. Percentages ofglial cell types in the ventral horn at various periods after RCA-60 injection Each figure represents the average value from 2 animals. Figures in parentheses are from the contralateral (non-injected side) ventral horn. Days after injection Oligodendrocytes Astrocytes Microglia 1 67-8 25 4 6-8 (65 8) (19 9) (13.3) 7 45.9 15-9 38-2 (67 9) (21-1) (11-3) 15 394 22-9 37-7 (65 8) (23 2) (1 -0) 30 47-1 27-6 25 3 (65 4) (22 8) (11-8) 60 63-2 19-2 17-6 (66 5) (24 5) (9 0)
Table 2. Corrected values ofpercentages ofglial cells in the ventral horn at various periods after RCA-60 injection Days after injection Oligodendrocytes Astrocytes Microglia (Control means) 66 2 22-3 11-3 1 66-2 24-8 6-6 7 66-2 22-9 55 1 15 66-2 38-5 63-3 30 66-2 38-8 35 6 60 66-2 20-1 18 4 microglia averaged 67 5%, 20-6% and 11 9% respectively. Following the injection of RCA-60 (Table 1), the proportion of microglia was drastically increased, so that by the seventh postoperative day the value was more than three times that of the normal or the contralateral side. The upsurge was maintained over a period of one week (up to the 15th day) and subsided gradually thereafter. Despite this, the proportion of microglia was maintained at a considerably higher level than the corresponding non- injected side in animals killed two months after the RCA-60 administration. Astrocytes, which constituted about one fifth of the total population, remained relatively unchanged throughout the period studied. The preponderant oligo- dendrocytes showed a corresponding decrease when the increase of microglial population was maximal in the early postoperative intervals. As will be noted later, our electron microscopic study revealed no evidence of degeneration or death of oligodendrocytes; it is therefore likely that their number remained constant and this is further supported by the observation that their percentages at one day and 60 days are similar (Table 1). The mean overall percentage of oligodendrocytes in the uninjected side (i.e. 66-2) may, then, be taken as normal. This figure must then be divided by the percentage of oligodendrocytes on the injected side and the corresponding percentages of astrocytes and microglia corrected by multiplying them by the resultant figures. The figures obtained are shown in Table 2. Neuroglial response to neuron injury 205
Figs. 4-5. For legends see page 206. 206 E. A. LING AND OTHERS Electron microscopy With the electron microscope, the normal ventral horn neurons displayed the usual structural features (Figs. 3,4) described by other authors (Rapoport & Stempak, 1968; Peters, Palay & Webster, 1976). Axon terminals containing round and flattened agranular vesicles were presynaptic to the somata of the neurons (Fig. 3). Widely scattered in the neuropil of the ventral horn were three kinds of neuroglial cells: astrocytes, microglia and oligodendrocytes with the latter cell type predominant (Fig. 4). Three and seven days after the injection of RCA-60 into the sciatic nerve, some of the ventral horn neurons underwent structural changes. The affected neurons were of the large type whose size ranged from 50 ,tm to 70,tm. The outline of the nucleus became irregular and crenated (Fig. 5). The cytoplasm of the soma and the radiating dendrites was pale and grainy due to the dispersion and disintegration of its organelles (Fig. 5). On closer examination, many vesicular profiles were present (Fig. 6). Cisternae of rough endoplasmic reticulum were sometimes arranged in spherical profiles (Fig. 6). Some degenerating neurons and their processes, however, assumed a dark appearance (Fig. 7). Vacuolation was obvious in their dense matrix (Fig. 7). In the same postoperative animals, a significant increase in the number of neuroglial cells, notably microglia, was observed in the region near the degenerating neurons (Fig. 5). Most of the microglial cells were distributed in the neuropil; only a few of them were seen occasionally in a perineuronal position (Figs. 5, 7). In what may be a more advanced stage of disintegration and degeneration in animals perfused 7 days after RCA injection, the neuronal soma was entirely sequestered by microglial cells (Fig. 8) while in some areas, the soma was invested by multilayered microglial cells and processes (Fig. 9). The microglial cells displayed all the characteristic structural features described by Mori & Leblond (1969). Their cytoplasm showed a flattened nucleus with coarse chromatic masses, and displayed organelles such as a Golgi apparatus, lipofuscin granules and small dense lysosome-like granules (Fig. 9). Cisternae of rough endoplasmic reticulum were long and stringy. There was no evidence of phagocytosis or pinocytosis of the neuronal cytoplasm by microglia. In rats with a longer survival period, i.e. 30 and 60 days after the RCA injection, the degenerating neuronal soma and its contained debris were completely disrupted resulting in an empty space walled by microglial cells (Fig. 10).
Fig. 4. A typical ventral horn neuron with its projecting dendrites (d) in the lower lumbar spinal cord of a normal rat. Five oligodendrocytes (0) are present in the neuropil. Lg, lipofuscin granules; N, nucleus; rER, rough endoplasmic reticulum. x 1425. Fig. 5. A pale degenerating ventral horn neuron in the lower lumbar spinal cord 7 days after RCA injection into the sciatic nerve. The nucleus (N) is irregular. Massive dense granules (arrows) are present in the cytoplasm especially in the perinuclear zone. Numerous neuroglial cells are seen in its vicinity. All the microglial cells, characterised by their coarse marginal chromatin masses and scanty cytoplasm, are numbered. Note that two of the microglial cells (1, 3) have already assumed a perineuronal position x 1400. Fig. 6. Portion of a pale degenerating neuron as in Fig. 5. Numerous vesicular profiles (V) are present near the extremely infolded nucleus (N). Cisternae of rough endoplasmic reticulum (rER), partly denuded of ribosomes, have a concentric arrangement. G, Golgi apparatus. x 10500. Fig. 7. A darkened degenerating neuron (N) with highly disorganised inclusions in the lower lumbar spinal cord. A group of eight microglial cells are in its vicinity. Seven days after RCA injection. x 1750. Fig. 8. A degenerating neuron at the stage of dissolution is sequestered entirely by several microglial cells and their processes (M). Arrows indicate some vesicular profiles that are probably swollen mitochondria undergoing degeneration. Ms, mitotic cell; 0, oligodendrocytes. Seven days after RCA injection. x 1750. Neuroglial response to neuron injury 207
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Figs. 6-8. For legends see page 206. 208 E. A. LING AND OTHERS
Figs. 9-13. For legends see page 210. Neuroglial response to neuron injury 209
Figs. 14-16. For legends see page 210. 210 E. A. LING AND OTHERS Following the injection of RCA-60, axon terminals forming synaptic contacts with degenerating neurons were gradually displaced by microglial processes. While most of the axon terminals that showed synaptic contacts with the dying neurons remained normal (Fig. 11), some appeared to undergo structural alteration and were engulfed by microglial processes (Fig. 12). In the neuropil, microglial cells were seen to phagocytose small myelinated axons (Fig. 13) especially in animals with longer survival times. In the large degenerating myelinated axons, however, microglial cells invaded the axon and insinuated themselves between the myelin sheath and the axoplasm. The axoplasm, containing swollen mitochondria, was engulfed by microglia. The activated microglial cells with their inclusion phagosomes eventually occupied the 'cavity' surrounded by the myelin sheath (Fig. 14). The majority of the microglial cells in rats with two months postoperative survival were filled with lamellated residual bodies, lipid droplets and lipofuscin granules (Fig. 15). Astrocytes undergoing mitotic division were occasionally observed in the neuropil (Fig. 16). Their cytoplasm was filled with bundles of microfilaments. They were not, however, seen to phagocytose degenerating profiles. There was no change in the structural features of oligodendrocytes throughout the period studied.
DISCUSSION The present study confirmed the selective destruction of the ventral horn neurons in the lumbosacral cord segment of rats following a single injection of RCA-60 into the sciatic nerve (Wiley & Oeltmann, 1986). Evidently, this was the result of a retrograde transport of the toxic lectin which poisoned the efferent neurons. The smaller neurons in the same area were not affected in the period studied. This would suggest that they are either interneurons or cells that are more resistant to the RCA injection. Unlike the common retrograde axonal reaction following a nerve transection (Kerns & Hinsman, 1973) or crush (Cova & Aldskogius, 1985), the degeneration of neurons induced by RCA-60 is rapid in onset, beginning three days after the administration of the drug. Furthermore, the degenerative process caused by RCA-60 is extremely drastic involving both the nucleus and the cytoplasmic organelles. A
Fig. 9. Microglial cells and their processes form a multilayered barrier between a degenerating neuron (s) and the neuropil (Np). The microglial cell has a flattened nucleus (N) with coarse chromatic masses. The Golgi apparatus (G) is well developed. Other organelles include lipofuscin granules (Lg) and cisternae of rough endoplasmic reticulum (rER). Arrows indicate lysosome-like granules. Seven days after RCA injection. x 14000. Fig. 10. A completely degenerated neuron is surrounded by multilayered microglial cells (M) and their processes (arrows). Thirty days after RCA injection. x 3500. Fig. 11. An axon terminal (A) is presynaptic to the soma (S) of a degenerating neuron. Note that the synaptic membrane densities are still present (arrows). M, mitochondria. Seven days after RCA injection. x 26250. Fig. 12. An axon terminal (A) appears to be surrounded and phagocytosed by two tongue-like microglial processes (arrows). m, mitochondria with disrupted cristae; S, degenerating neuronal soma. Seven days after RCA injection. x 26250. Fig. 13. A disorganised and degenerating myelinated axon (A) is phagocytosed by a microglial cell (A). Two months after RCA injection. x 8750. Fig. 14. Four activated microglial (AM) cells with their pseudopodial processes (arrows) penetrating a large degenerating myelinated axon (A). Seven days after RCA injection. x 4375. Fig. 15. A typical microglial cell in the ventral horn of the lumbosacral cord 2 months after RCA injection. The cytoplasm is filled with liposfuscin granules (Lg) and lamellated residual bodies (LR). x 14000. Fig. 16. An astrocyte undergoing mitotic division. Its pale cytoplasm shows bundles of microfilaments (/). Seven days after RCA injection. x 3000. Neuroglial response to neuron injury 211 striking change is the dissolution of the cisternae of rough endoplasmic reticulum. This finding supports the proposition of MacConnel, Eurman & Kaplan (1982) that ricin acts by inhibiting neuronal protein synthesis. An interesting feature observed in the present study is the condensation and darkening of some cells including their dendritic processes. A similar darkening phenomenon was also observed in some RCA-poisoned neurons in the dorsal motor nucleus in rats (Ling & Leong, 1987) and in several regions in the central nervous system following axonal injury (Barron, 1975). It may also occur artefactually as reported by Cammermeyer (1962) and Mugnaini (1965). The significance of darkened neurons needs further elucidation. It is remarkable that in the initial stage of degeneration, axon terminals making synaptic contacts with neurons appear structurally unchanged. With time, however, they become displaced by invading microglial cells. A similar phenomenon has also been described in axotomised facial motoneurons (Blinzinger & Kreutzberg, 1968) and spinal motoneurons of the lower lumbar cord (Kerns & Hinsman, 1973). On very rare occasions, the displaced axon terminals appear to be phagocytosed by microglial cells. In this connection, the terminals with their contained synaptic vesicles are disorganised and could be assumed to be non-functional. The majority of the terminals, however, retain their normal features and are pushed into the neuropil, far removed from the poisoned neuronal somata by the intervening microglial processes. The most dramatic response following degeneration of the ventral horn neurons induced by the injected RCA-60 is shown by the reaction of the microglial cells. Since such a drastic response has not been observed in similar material and time interval following an ordinary sciatic neurectomy (Kerns & Hinsman, 1973), the reaction could be attributable to the toxic ricin. This is manifested by their rapid increase in number in the vicinity of degenerating neurons. Three and seven days following the administration of RCA-60, numerous microglial cells assume a perineuronal position rather similar to the 'microglial cluster' described by Streit & Kreutzberg (1988). However, there is no evidence of a direct pinocytosis or phagocytosis of neuronal somata undergoing dissolution. Following the displacement of the axonal terminals, the degenerating neuronal soma is sequestered by the multilayered microglial cells and their processes. It is possible that such a configuration may be a protective mechanism preventing the spread of toxic substance that could have leaked out from the degenerating neurons. It is unlikely that the RCA-60 taken up by the efferent neurons would exert a transneuronal effect on the axon terminals making synaptic contacts with them nor with the adjacent smaller neurons, some ofwhich could be interneurons. The presence of a few occasional terminals which appear to be phagocytosed by microglial cells suggests that they have become non-functional following the death of the postsynaptic neurons. While there is no evidence to indicate the involvement of microglial cells in the phagocytosis of the somata of degenerating neurons, it is clear that microglial cells are definitely involved in the phagocytosis of degenerating myelinated axons, particularly in animals with longer survival. This supports the view of the phagocytic nature of microglial cells (Sjostrand, 1971; Kerns & Hinsman, 1973; Torvik, 1975; Streit & Kreutzberg, 1988), although their phagocytic capability has been questioned by some authors (Fujita & Kitamura, 1976; Oehmichen, 1978). Most of the degenerating elements have been cleared by two months after the RCA injection and this appears to coincide with the decline of the microglial population. Most of the microglial cells observed at this postoperative interval are loaded with residual bodies in the form of lipofuscin granules, suggesting that they have completed their scavenger function. 212 E. A. LING AND OTHERS The upsurge of microglial cells at the site of neuronal degeneration may be due to a reactive proliferation of local microglia as proposed by Kerns & Hinsman (1973). In an earlier investigation, using a radioautography method, Sjostrand (1971) also showed an active proliferation of local neuroglial cells in the hypoglossal nucleus following a crush of the hypoglossal nerve. So far, we have no evidence of any infiltration of mononuclear cells into the site of degeneration as observed in the dorsal motor nucleus of the vagus nerve following intraneural injection of a similar toxin (Ling & Leong, 1987, 1988). It may be mentioned in this connection that in a pilot histochemical study of the present materials, the microglial cells encircling the degenerating neurons were negatively stained by the reaction for non-specific esterase. These observations therefore tend to support the view of Streit & Kreutzberg (1988) that the increase in the number of microglia is probably the result of proliferation of local microglia. It would seem therefore that neuronal death or degeneration at different sites in the nervous tissue would elicit a response from different sources of neural macrophages. Since there is no indication of any dead or degenerating oligodendrocytes in the postsurvival period studied, the apparent decline in their percentage population (see Table 1) following the RCA injection is the result of the increase in the relative number of other glial cells. Thus, there is a significant increase in the proportion of astrocytes and microglia between 7 and 30 days after RCA injection (Table 2). Table 2 shows a two-phase glial response: the first is an increase in microglia peaking at 7-15 days, and then gradually decreasing; this is followed by a second, smaller increase in astrocytes, peaking at 15-30 days, and this also declines thereafter. The increase in astrocytes may explain the occurrence of mitotic astrocytes seen at 7 days after RCA injection. Their increase may be related to scar formation (Peters et al, 1976) following neuronal degeneration. Although considered to be potential phagocytes (Ling, Wong, Yick & Leong, 1986), there is no evidence that astrocytes take part actively in the phagocytosis of the RCA-poisoned neurons.
SUMMARY The present study has shown the selective destruction of large ventral horn neurons in the lumbosacral cord segments following a single injection of RCA-60 into the sciatic nerve. The neurons appeared to undergo structural alteration beginning 3 days after the RCA application. In the postoperative period extending from 1 to 60 days, degeneration of neurons was progressive and irreversible and this elicited a rapid increase in the number of microglial cells. They were most numerous in the 7 days postoperative animals. The massive microglial cells penetrated the neuropil and appeared to strip off the axon terminals from the postsynaptic somata. Occasional axon terminals were phagocytosed by microglia. The numerous microglial cells often formed a multilayered 'barrier' encircling the somata of the RCA-poisoned neurons which eventually became totally disorganised. It is postulated that in the course of neuronal degeneration induced by RCA, microglial cells serve to prevent the leakage or diffusion of the toxic lectin from the neuronal somata into the neighbouring neuropil. They also function as scavenger cells in the removal of degenerating myelinated axons in the longer surviving rats. Oligodendrocytes do not appear to react actively to the degeneration process. However, astrocytes showed a significant increase in the 7 and 15 day postoperative rats and this coincided with the presence of mitotic astrocytes in the same period. Neuroglial response to neuron injury 213 The present study was supported by a research grant (Project No. RP880310) from the National University of Singapore. The financial support from the National Science Council, Taiwan (Research Grant NSC-77-0412-B-002-127) is gratefully acknow- ledged.
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