Neuroglial Response to Neuron Injury. a Study Using Intraneural Injection of Ricinus Communis Agglutinin-60
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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 - s 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.