HistochemicaI Journal 16, 165-178 (1984)

Neuronal and oligodendrocytic response to cortical injury: ultrastructural and cytochemical changes S. Y. A . A L - A L I ~ a n d N . R O B I N S O N

2

1Department of Anatomy, University of Glasgow, Glasgow, G12 8QQ 2Department of Anatomy, The London Hospital Medical College, London, E1 2AD, U.K. Received 11 March 1983 and in revised form 13 June 1983

SumlTlary A needle wound was made in the adult rat cerebral cortex. Responses of neurons and oligodendrocytes at the site of injury were followed over a period of 450 days and correlations made between morphological and enzyme cytochemical changes to clarify some phenomena previously unresolved. Evidence from acid phosphatase activity in degenerating neurons showed no increase in the number of cytochemically stained lysosomal profiles nor changes in the subcellular localization of the acid phosphatase reaction product. Our observations indicated that the majority of dying neurons were not digested by their own acid phosphatase 'autodigestion' but by the process of heterodigestion. The time-course study revealed that not all the traumatized neurons were eliminated but some persisted permanently in an attenuated 'atrophic' state. The atrophic neurons were small in size with low cytoplasmic-nuclear ratios and exhibited low levels of glucose-6-phosphatase and cytochrome oxidase activities. The acid phosphatase activity was slightly increased as evidenced by cytochemically stained hypertrophic Golgi cisternae and a slight increase in the number of lysosomes. The low level of enzyme activities concerned with carbohydrate metabolism reflected the low metabolic activity in atrophic neurons whilst an increase in Golgi-lysosomaI enzyme activity suggested some anabolic process necessary for their survival. Oligodendrocytes displayed only minor changes in morphology, and their glucose6-phosphatase and cytochrome oxidase activities were normal, suggesting that these cells have little or no involvement in the repair of a cerebral wound. The absence of significant changes in lysosomal acid phosphatase activity indicated a minimal role, if any, of oligodendrocytes in the process of phagocytosis. Introduction Descriptions of the cytological r e s p o n s e of central n e r v o u s tissue to a surgical w o u n d have b e e n r e p o r t e d sporadically for several decades (Penfield & Rio-Hortega, 1932; 0018-2214/84 $03.00 + .12

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Imamoto & Leblond, 1977; Bernstein etaI., 1978; A1-Ali & Robinson, 1978a, b). However, there are areas where either the information is incomplete or the results are conflicting. This is particularly true for the process of digestion, the fate of traumatized neurons and the response of oligodendrocytes to the injury. Two viewpoints expressed on the digestion of traumatized neurons are that they are digested and eliminated by their o w n hydrolytic enzymes (McKeever & Balentine, 1973; Decker, 1978) or by hydrolytic enzymes of other cells (Glover, 1982). On the fate of the neurons, some reports indicate that all the affected neurons in the central nervous system degenerate and die (Bernstein, 1967; Persson, 1976), whilst other reports suggest a partial reversibility of morphological changes (Svendgaard et al., 1976; Barron & Dentinger, 1979) with evidence to support this from the growth of nerve cell transplants in mammalian brain (Raisman et al., 1980). Some reports on the reaction of oligodendrocytes in response to brain injury describe a major role for these cells in phagocytosis and w o u n d repair (Persson, 1976; Herndon et al., 1977; Nadler et al., 1980) but others give no such support to a major role of oligodendrocytes in w o u n d repair (Schultz & Willey, 1976; Imamoto & Leblond, 1977). In this study, the response of neurons and oligodendrocytes have been examined in an attempt to answer the following questions: Do all the traumatized cortical neurons degenerate and then become eliminated or do some take an attenuated state and remain permanently in an altered form? Do the degenerating neurons eventually die and then get digested by their o w n acid phosphatase enzyme (autodigestion) or by acid phosphatase of other cells (heterodigestion)? Does the acid phosphatase activity remain confined within membrane-bound Golgi-lysosomal organelles or do the enzyme reactions become diffuse within the cytoplasm of the degenerating neuron? Does the trauma to the cortical neurons stimulate oligodendrocytes to become an active form which could be detected morphologically and shown cytochemically to exist in a similar manner to reactive astrocytes? Are oligodendrocytes involved in the process of phagocytosis? If so, could this be shown by acid phosphatase activity? A needle w o u n d was used to study the neuronal and oligodendrocytic response since the lesion is simple, remains localized, does not produce neurological symptoms and is used in experimental animals and in some routine neurosurgical procedures. The enzymes studied were acid phosphatase, glucose-6-phosphatase and cytochrome oxidase, all of known ultrastructural distribution in normal brain (Brunk & Ericsson, 1972; A1-Ali & Robinson, 1979a, 1981). Some of the results have been presented in a preliminary form (A1-Ali & Robinson, 1982).

Materials and methods Adult male Sprague-Dawley rats, weighing 250 g, were anaesthetized with chloral hydrate and secured in a stereotaxic instrument. Under asceptic conditions a stab wound was produced in the cortex at a point i mm anterior to the coronal suture and 1.5 mm to the left of the median sagittal suture using a 25-gauge hypodermic needle. The needle was inserted into the cortex to the level of

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the corpus callosum. The animals showed no post-operative neurological or functional disturbances. They were allowed to survive for periods of 1, 2, 3, 5, 7, 12, 14, 30, 60, 120, 240 and 450 days post-operation. Brains were fixed by vascular perfusion with a mixture of formaldehyde and glutaraldehyde. Coronal slices of the cerebral cortex were sectioned at 40 pm with an Oxford vibratome at the site of injury. The enzymes examined for reactive changes and markers for cell organelles were acid phosphatase for lysosomes, glucose-6-phosphatase for the granular endoplasmic reticulum using the lead method, and cytochrome oxidase for mitochondria usil~,g the diaminobenzidine (DAB) method. The opposite 'unoperated' cortex of experimental animals and sham experiments served as controls. Light and electron microscopical techniques for the morphological experiments and enzyme cytochemistry have been described previously (A1-Ali & Robinson, 1979a; 1981; 1982a). A MOP system (AM 02) was used to measure the areas (in #m 2) of neuronal cytoplasm and nucleus on semi-thin plastic sections for quantitative information.

Results

The response of cortical neurons and oligodendrocytes to the needle w o u n d was localized within a narrow band of tissue parallel to the needle path (Fig. la). In the unoperated and sham hemispheres no changes from the normal morphology and cytochemistry were apparent.

Morphological changes In the immediate vicinity of the wound, neurons became necrotic and were removed by phagocytes at 5-7 days post-operation. Neurons further removed from the w o u n d showed alterations which varied from a mild chromatolysis to an advanced stage of degeneration. The chromatolytic changes consisted of a decrease in electron density of the nucleus and cytoplasm (swollen, pale neurons), depletion of the granular endoplasmic reticulum, ribosomes and mitochondria, and the cytoplasm appearing vacuolated and swollen (Fig. 2). Another type of degenerating neuron, sometimes observed, exhibited increased electron density of the nucleus and cytoplasm (shrunken, dark neurons), extensive folding of its nuclear envelope and the cytoplasm appeared granular (Fig. 3). Phagocytes were frequently seen abutting or surrounding the degenerating neurons (Figs. 2, 3). At 14 days post-operation, the w o u n d site became permanently separated from the cortex by an astroglia-connective tissue barrier. The period before the formation of the barrier was referred to as the 'early period' and after that as the 'later period'. Some of the altered neurons continued to degenerate and were removed by phagocytes between 14-30 days post-operation. Other neurons exhibiting permanent alterations were seen throughout the remainder of the 450 days experimental period in the region adjacent to the w o u n d barrier. Alterations in these surviving neurons consisted of atrophic changes: the neurons were smaller than normal, displayed smooth contours and a reduction in the cytoplasmic-nuclear areas and ratio (Fig. lb, Table 1).. Consistently invaginated nuclei showed chromatin condensations associated with the nuclear envelope and nucleoli. The cytoplasm displayed a low electron density,.

|a

Fig. 1. (a) Diagram showing the position of the needle wound on the surface of the left cerebral hemisphere and in a coronal section. (b) 1-2/~m plastic section showing the wound site at 450 days post operation. Neurons adjacent to the wound barrier showing atrophic changes such as small cell size, round and smooth contours, with pale and thin rim of cytoplasm (arrows); W, needle wound, x 575.

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Fig. 2. Swollen neuron showing acid phosphatase reaction product discretely localized in Che lysosomes (Ly) and Golgi cisternae (G) at 5 days post-operation; Ph, phagocyte. Lightly stained with lead citrate, x 11 300.

contained few mitochondria, short granular endoplasmic reticulum and few ribosomes; however, some atrophic neurons showed prominent arrays of Golgi cisternae (Fig. 4)~ Oligodendrocytes in the immediate vicinity of the wound, like the neurons, became necrotic and were removed by phagocytes between 5-7 days post-operation. Further from the wound, and within the area of the neuronal chromatolysis, the ultrastructural morphology of oligodendrocytes remained unchanged throughout the 450 day period. The cytoplasm and processes appeared well preserved within tissue composed of numerous degenerated myelin sheaths and axons. However, a multilayered sheath surrounding the oligodendrocytes, probably composed of aberrant myelin, was frequently seen during 14-30 days post-operation (Fig. 5).

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Table 1. Changes in areas (pm 2) of perikaryal cytoplasm and nucleus of surviving atrophic neurons as compared with adjacent normal neurons in the cerebral cortex following a needle wound injury.

Measurements

Normal neurons

Atrophic neurons

Number of cells measured Area of 100 neuronal perikarya (/lm 2) Area of perikaryal cytoplasm of 100 cells (pm 2) Area of nuclei of 100 cells (~m 2) Cytoplasmic-nuclear ratio

100 14 252 7464 6788 1.10

100 8933 3810 5123 0.74

Areas of atrophic neurons

Reduction in the area of neuronal perikarya of atrophic neurons as compared with normal neurons Reduction in the area of perikaryal cytoplasm of atrophic neurons as compared with normal neurons Reduction in the area of nuclei of atrophic neurons as compared with normal neurons Reduction in the cytoplasmic-nuclear ratio of atrophic neurons as compared with normal neurons

Percentage changes in atrophic neurons

37.3% 48.9% 24.5% 32.7%

Fig. 3. Shrunken (dark) neuron exhibiting discretely localized acid phosphatase reaction product in lysosomes (Ly) and Golgi cisternae (G) at 5 days post-operation; Ph, phagocytes. Lightly stained with lead citrate, x 9700. Fig. 4. (a) Normal neuron from the control 'unoperated' cerebral cortex; the cytoplasm is rich in cell organelles; granular endoplasmic reticulum (ER); lysosomes (Ly); Golgi complexes (G). x 6 500. (b) Atrophic neuron exhibiting nuclear invagination by a deep cytoplasmic furrow; the thin rim of the cytoplasm containing few cell organelles but prominent Golgi complexes (G) at 240 days post-operation, x 12 300. Fig. 5. Oligodendrocyte surrounded by a multilayered sheath, probably composed of aberrant myelin at 30 days post-operation, x 12 900. Fig. 6. Acid phosphatase reaction product in prominent Golgi cisternae (G) and lysosomes (Ly) of atrophic neuron at 30 days post-operation. Lightly stained with lead citrate, x 20 400. Fig. 7. Acid phosphatase reaction product in the Golgi cisternae (G) and lysosomes (Ly) of normal neuron from the control 'non-operated' cerebral cortex. Lightly stained with lead citrate, x 12 900. Fig. 8. Cytochrome oxidase activity in atrophic neuron, the surviving mitochondria showing the enzyme reaction product at 14 days post-operation. No counterstain, x 17 400. Fig. 9. Glucose-6-phosphatase activity is markedly diminished or absent from the nuclear envelope and the remaining cisternae of the granular endoplasmic reticulum of atrophic neuron (a) at 30 days post-operation, as compared with normal neuron of the cerebral cortex (b). The lead precipitate on lysosomes is related to the interfering acid phosphatase activity. Lightly stained with lead citrate. (a) x 10 000. (b) x 34 000.

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CytochemicaI changes The cytochemistry of neurons in normal and traumatized tissue showed elements o:f lysosomes exhibiting a discrete acid phosphatase reaction product localized exclusively within membrane-bound Golgi-lysosomal organelles. In traumatized neurons there was no increase in the number of cytochemically stained Iysosomal profiles and no changes in localization of the acid phosphatase reaction product could be detected in pale or dark degenerating neurons in the early period (Figs. 2, 3). In the later period, surviving atrophic neurons exhibited acid phosphatase within an increased number of lysosomes and within prominent arrays of Golgi cisternae w h e n compared with normal neurons (Figs. 6, 7). Cytochrome oxidase activity, prominent in the mitochondria of normal neurons (A1-Ali & Robinson, 1979a), was substantially reduced during the early period and was present in a few surviving mitochondria of atrophic neurons at the later period (Fig. 8). Glucose-6-phosphatase activity, intense and well localized in normal granular endoplasmic reticulum, was substantially reduced or absent in early degenerating and in later surviving atrophic neurons, the nuclear envelope and remaining cisternae of the granular endoplasmic reticulum showed little or no reaction compared to the normal (Fig. 9a, b). Throughout the 450 days experimental period, oligodendrocytes showed no changes in the lysosomal acid phosphatase activity nor increase in lysosomal profiles, the reaction resembling normal oligodendrocytes (Fig. 10). Cytochrome oxidase and glucose-6-phosphatase activities remained unchanged in oligodendrocytes (Fig. 11). Discussion

A major difficulty encountered in the study of fine structure of the central nervous system is tissue preservation, since rapid post-mortem changes occur with inadequate preservation w h e n artefactual changes can be erroneously interpreted as a pathological reaction. This problem is augmented in the study of enzyme cytochemistry; hence, effects were directed towards improvements in the techniques until a consistent adequate fine structural preservation for enzyme cytochemistry was obtained in normal cortex before the techniques were applied to traumatized cortex (A1-Ali & Robinson, 1979a, b, 1981). The evidence for efficiency of a fixation technique is the preservation of astrocytes, since they are the cell type most vulnerable to fixation artefacts in central nervous tissue (Persson, 1976); this was achieved in normal and then applied to traumatized cortex (A1-Ali & Robinson, 1982a). The tissue for the present study was Fig. 10. Acid phosphatase activity in oligodendrocyte, the enzyme reaction product is in lysosomes and some Golgi cisternae at 5 days post-operation. Lightly stained with lead citrate, x 20 000. Fig. 11. Glucose-6-phosphatase activity in oligodendrocyte, the enzyme reaction product is in the nuclear envelope and cist~rnae of the granular endoplasmic reticulum at 14 days post-operation. Lightly stained with lead citrate, x 28 000.

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from the same material used for the study of astrocytes (A1-Ali & Robinson, 1982a). In addition, various controls were applied to confirm the authenticity of neuronal and oligodendrocytic reactions to the injury. Two types of structural changes that occurred in neurons during the early period were an increase or decrease in the electron density referred to as 'dark' or 'pale' neurons respectively. Pale neurons were more frequently seen than dark neurons; a decrease in the electron density of neurons was also observed after axotomy and attributed to cellular swelling (Grafstein, 1975). Dark neurons have been the subject of controversy as to whether the changes were due to fixation artefact (Paljarvi et aL, 1979) or to a reaction due to severe trauma, signalling inevitable cell death (Aldskogius & Arvidsson, 1978; Nadler et al., 1980). The detection of dark neurons during the early period only, absence of these ceils in the controls and the presence of phagocytes abutting the dark neurons, presumably preparing for their removal, indicated that the structural changes occurred before death of the animal. It was concluded that the increase in the electron density was more likely to be a reaction due to direct damage to cell soma or its major processes or due to a disruption to their blood microcirculation (Persson, 1976). Observations on acid phosphatase activity during the early period showed no evidence that degenerating neurons complete their own digestion, since there was no increase in the number of cytochemically stained lysosomal profiles and no intracellular release of free reaction product of acid phosphatase in degenerating neurons. These observations give substance to an earlier suggestion that degenerating neurons were digested by acid phosphatase of brain phagocytes (A1-Ali & Robinson, 1982b). However, the possibility of some autodigestion cannot be totally excluded; it might have occurred within a few neurons or for a very short period that went undetected, or the sensitivity of the acid phosphatase cytochemical technique may have been above the threshold of autodigestion. Later periods witnessed a persistence of some neurons in an atrophic form, providing evidence that some neurons in the central nervous system change their course of degeneration and assume an atrophic state rather than suffer cell death and elimination after a stab wound. Failure to identify atrophic neurons in earlier studies using paraffin and frozen sections was due to the fact that the atrophic neurons were small in size and did not take a histological stain (Egan et al., 1977). In this study, the use of plastic sections improved the optical resolution and made the identification of atrophic neurons possible and this was confirmed by electron microscopy. The quantitative and morphological study showed a significant reduction in the perikaryal cytoplasm and cellular organelles. This correlated well with the enzyme cytochemistry which gave new evidence that atrophic neurons were altered metabolically as well as morphologically; the metabolic changes witnessed in the diminished glucose-6-phosphatase and cytochrome oxidase activities reflecting the low level of carbohydrate metabolism. The significance of a slight increase in the acid phosphatase-positive Gotgi-lysosomal profiles suggests occurrence of anab0!ic processes which made the survival of some neurons possible, albeit with attenuated metabolism.

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Cortical neurons responded differently to the needle w o u n d injury, the differences due possibly to the severity of the damage or to the occurrence of more than one kind of reactive process in the altered neurons, the balance between the catabolic and anabolic activities presumably determined the enzyme cytochemical and morphological changes. The response of oligodendrocytes to injury in adult central nervous tissue has no~ been clearly understood and has remained controversial (see Introduction). In this study, there were no major changes in their morphology or enzyme cytochemistry in sharp contrast to the dramatic changes occurring in astrocytes (A1-Ali & Robinson, 1982a). The absence of apparent changes in glucose-6-phosphatase and cytochrome oxidase activities compared with normal oligodendrocytes suggested no alterations in their metabolic activities. Absence of detectable changes in acid phosphatase activity indicated that involvement of oligodendrocytes in the process of phagocytosis is unlikely, at least not without losing their identification of being oligodendrocytes according to the morphological criteria of Peters et al. (1976). Therefore, it was concluded that oligodendrocytes played a minimal role during w o u n d repair in adult rat cerebral cortex. However, it remains of interest as to w h y the oligodendrocytes appeared well preserved morphologically and cytochemically amidst much traumatized neural tissue.

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