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A combined light and electron microscope study of early anoxic-ischaemic cell change in rat brain
BRAIN RESEARCH
193
A COMBINED L I G H T AND ELECTRON MICROSCOPE STUDY OF EARLY ANOXIC-ISCHAEM1C CELL C H A N G E IN RAT BRAIN
S. M.
McGEE-RUSSELL*, A. W. BROWN ANDJ. B. BRIERLEY
Virus Research Unit and Neuropsychiatric Research Unit, Medical Research Council Laboratories, Carshalton, Surrey (Great Britain)
(Accepted November 21st, 1969)
INTRODUCTION Microvacuolation (MV) of the cytoplasm was described as the earliest neuronal alteration resulting from anoxia-ischaemia in the ipsilateral cerebral hemisphere of the rat 'Levine preparation'. It was demonstrable with the light microscope in conventionally stained paraffin sections of brains that had been fixed by perfusionl,L Microvacuolation was succeeded by ischaemic cell change (ICC), ischaemic cell change with incrustations and cell loss. Microvacuolation was recognizable after exposures to nitrogen of 30 and 20 min. In the present study a survival of 1 h after 40 rain exposure to nitrogen was chosen in order to observe transitional stages between MV and ICC. The ultrastructural examination was restricted to zone hl (CA1) of the hippocampus because of its high neuronal density and its known vulnerability to anoxiaischaemia. The purpose of the present investigation was to correlate the light microscopical appearances of microvacuolated neurones in thin large-area plastic sections 3,8 with those obtained with the electron microscope and to define their ultrastructural features, origin and associated subcellular changes. MATERIALSAND METHODS The experimental procedures have been describedl, 2. Four rats (White Wistar, Carshalton strain, weight 150 g) were exposed to nitrogen (5 litres/min) 0.25-3 h after application of the 'artery clasp' to the right common carotid artery. When apnoea occurred mechanical ventilation was employed until spontaneous respiration was resumed. The cycle, quiescence, hyperpnoea,
* Present address: Department of Biological Sciences, State University of New York at Albany, Albany, N.Y. 12203, U.S.A.
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convulsion, apnoea and mechanical ventilation, was repeated (average 10 times. range 4-17) during the 40 min. One hour later, under ether anaesthesia, the 'artery clasp' was removed and the brain was perfused with glutaraldehyde (6.5 ~ in cacodylate buffer, p H 7.4, minimum 250 ml during 20 rain) without initial perfusion with saline. The brain was left in situ for 2 h and was then removed and stored in the fixative at 4°C overnight. The forebrain was cut coronally through the pituitary stalk and a 1-2 m m slice posterior to this plane was embedded in plastic. The next posterior slice was embedded in Paraplast (Shandon Scientific Co. Ltd.) for light microscopical study. The EM slice was left in cacodylate buffer at 4°C for 24 h and post-fixed in osmium-cacodylate fixative (1% OsO4 in cacodylate buffer pH 7.4) at 4°C for 18 h. The slice was embedded in Epon 812 (5A:5B, LuftT). Sections were cut on a Porter Blum M T 1 ultra-microtome using sharpened steel edges to obtain sections 2-4/~m thick. These were stained with toluidine blue or cresyl fast violet at 60°C and were mounted in Epon on glass slides. Regions containing altered neurones in these sections were prepared as 'mesa pyramids' (1 mm × 1 mm) on the cut surfaces of the original blocks so that ultra-thin sections could be taken for electron microscopy. These were stained with 2 % uranyl acetate, lead citrate (animal IV only) and examined with an AEI EM 6 electron microscope at accelerating voltages of 60 and 40 kV, with 400 # m condenser apertures and 25/zm objective apertures. Paraffin sections (7 and t2 #m) were stained with cresyl fast violet, toluidine blue, Luxol fast blue and cresyl fast violet and with haematoxylin and eosin. The brains of 4 normal rats were fixed by perfusion with glutaraldehyde, removed 2-3 h later and all subsequent stages were carried out as above.
RESULTS
Controls
In the 4 animals the brain was uniformly tanned but appreciably shrunken. In paraffin and plastic sections (Fig. I A and B) staining was uniform and there was no evidence of artefact.
Fig. 1. A, Control rat. Hippocampus (hl). Paraffin section showing selective staining of neurones, endothelial cells and the absence of artefact. Cresyl fast violet, x 780. B, Control rat. Hippocampus (h!). Epon-embedded. Section (3/~m) showing general staining of all cellular elements and the absence of artifact. Cresyl fast violet, x 780. C, 'Levine preparation', ipsilateral hippocampus (hi). Animal IV. Paraffin section showing pyramidal cells containing microvacuoles separated by strongly basophilic cytoplasm. Toluidine blue. x 800. D, As for part C. Animal III. Epon-embedded. Section (3 #m) showing microvacuoles in neuronal cytoplasm and conspicuous perineuronal and perivascular spaces. Cresyl fast violet, x 800. E, As for part C. Animal III. 1 ~m section from 'mesa pyramid' showing microvacuoles evenly distributed and extending into the apical dendrite. Toluidine blue. x 1460. Brain Research, 20 (1970) 193-200
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Brain Research, 20 (1970) 193-200
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S.M.
MCGEE-RUSSELL e[ ol.
Experimental animals Macroscopic In all 4 animals slight prominence of the inferior cerebellar vermis was evidence of minimal brain swelling. In coronal slices the ipsilateral neocortex was widened in all, least in animal i and most in animal lII.
Microscopic (a) Paraffin sections. Altered neurones were seen in the ipsilateral neocortex of all 4 animals but MV and ICC were seen in the hippocampus of only animals 1II and IV and in these two animals this structure was submitted to ultrastructural study. In the pyramidal cells of the hippocampus microvacuoles appeared as apparently empty circular spaces within a strongly basophilic cytoplasm (Fig. 1C). The nucleus was either normal or slightly shrunken, the nucleolus appeared normal and the cell was of normal size. (b) Plastic sections. Within the hippocampus (hl) there were no normal neurones. In a minority the cytoplasm showed a slight increase in staining intensity and contained a few microvacuoles; the nucleus and nucleolus were unaltered. The majority of neurones were of normal size and their darkly stained cytoplasm was virtually filled with microvacuoles (Fig. I D). A few microvacuoles could be seen in occasional intensely stained shrunken cells. Microvacuoles were usually circular but occasionally oval, they appeared to be empty and their diameters were 0.16-2.5 #m. They were evenly distributed in the cytoplasm and were often seen in the proximal portion of the apical dendrite (Fig. 1E). Increased cytoplasmic staining was associated with a reduced number of microvacuoles, shrinkage of the cell and a triangular darkly-staining nucleus.
Fig. 2. As for Fig. 1C. Animal IV. Low-power electron micrograph showing 2 neurones (n) containing microvacuoles (arrows). The cytoplasm is shrunken and the nucleus (centre) triangular. Swollen astrocytic processes (ap) surround the neurones and a blood vessel (bv). × 3375.
Brain Research, 20 (1970) 193-200
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Fig. 3. As for Fig. IC. Animal III. The microvacuoles (mv) are bounded by double membranes and some contain remnants of membranous material. Note the dilatation of the tubules and cisternae of the endoplasmic reticulum (arrows) and also occasional expansion of the nuclear membrane cisternae. There appears to be an accumulation of free ribosomes and ribosomal rosettes accompanied by an increase in the cytoplasmic matrix density. × 24,250.
Brain Research, 20 (1970) 193-200
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The conspicuous separation of the damaged neurones was due to wide perineuronal spaces, subdivided by trabeculae and either empty or containing a few cytoplasmic remnants. Similar spaces surrounded small blood vessels. Much smaller spaces gave a 'loosened' appearance to the adjacent neuropil. (e) Electron microscopy. Fig. 2 illustrates a typical microvacuolated cell. Each microvacuole consisted of an electron-lucent space bounded by two unit membranes (Figs. 3 and 4). Within the space there was often a small amount of high contrast material of ill-defined character probably representing remnants ofcristae. The majority of microvacuolated cells were surrounded by the swollen processes of astrocytes (Fig. 2) which corresponded to the perineuronal spaces seen with the light microscope. Microvacuolated cells were characterized by the absence of normal mitochondria but the presence of a double membrane around microvacuoles and the nature of their contents indicated their origin from mitochondria. Some dilatation of the tubules, vesicles and cisternae of the endoplasmic reticulum was also observed (Fig. 3). The material within the expanded membranes was of low electron density. There was an aggregation of free ribosomes and ribosomal rosettes together with an increased density of the cytoplasmic matrix (corresponding to the intensely basophilic microvacuolated neurones seen with the light microscope; Fig. IC-E).
Fig. 4. As for Fig. 1C. Animal III. High-power electron micrograph showing two swollen mitochondria containing remnants of cristae, x 68,300. Brain Research, 20 (1970) 193-200
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Within microvacuolated neurones, some nuclei were normal while others showed an aggregation of nuclear material sometimes accompanied by widening of the space between the nuclear membranes (Fig. 3). The majority of mitochondria in the swollen astrocytic processes and also in the adjacent neuropil appeared normal. DISCUSSION In the present investigation, the 'large area sectioning method' (refs. 3, 4) has facilitated the survey and detailed study of control and experimental brain tissue. Evidently the apparently empty microvacuoles seen in both paraffin and plastic sections are accounted for in most instances by swollen mitochondria and, in a minority, by dilatations within the endoplasmic reticulum. Hitherto, ultrastructural studies of the time course of anoxic-ischaemic alterations have been limited by either the employment of immersion-fixation6 or inadequate perfusion-fixation4,5,9. Hillse, using the Levine preparation, described swelling of astrocytic processes after 1.5 h but no alteration in neurone or capillary endothelium until 3 h. Hager et al. 4, Hager 5 and Scholz and Hager 9, using perfusion-fixation with osmic acid, studied the fine structure of the neocortex of the Syrian hamster killed immediately after repeated asphyxiation during a 12-15 min exposure to nitrogen. There was no parallel light microscopic study. Swelling of mitochondria was seen in neuronal cytoplasm of reduced rather than increased electron density. Such appearances are an artefact arising from inadequate perfusion-fixation, even in the brain of a normal animal (Brown and Brierley, unpublished observations). In the present study, the internal derangement of mitochondria implies some impairment of function of their oxidative enzymes. The parallel expansion of astrocytic processes implies that it has yet to be decided whether astrocytic or neuronal alterations are the first consequence of anoxia-ischaemia. SUMMARY Anoxic-ischaemic neuronal damage was studied by a combined light and electron microscope study of the brains of 4 rats exposed to nitrogen intermittently during a period of 40 min after interruption of blood flow in the right common carotid artery ('Levine preparation'). The animals were killed by perfusion-fixation with glutaraldehyde after survival of 1 h (measured from the end of exposure to nitrogen). Microvacuolation, the earliest alteration, was abundant in the ipsilateral hippocampi (hl) of two animals. The electron microscope study was restricted to this region. Microvacuoles in paraffin and plastic sections (light microscope) appeared as apparently empty circular spaces (0.16-2.5/zm in diameter) in the cytoplasm. At the ultrastructural level most microvacuoles were seen to be swollen mitochondria retaining their double membranes despite progressive disruption of their internal structure. Brain Research, 20 (1970) 193-200
s.M. MCGEE-RUSSELLet al.
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Di l at at i o n s o f the tubules, vesicles and cisternae o f the e n d o p l a s m i c reticulum were also observed. T h e r e was an associated a p p a r e n t increase in r i b o so m es and in c y t o p l a s m i c m a t r i x density. The d a m a g e d n e u r o n e s were s u r r o u n d e d by swollen astrocytic processes.
REFERENCES 1 BROWN,A. W., AND BRIERLEY, J. B., Evidence for early anoxic-ischaemic cell damage in the rat brain, Experientia (Basel), 22 (1966) 546-547. 2 BROWN, A. W., AND BRIERLEY, J. B., The nature distribution and earliest stages of anoxic-ischaemic nerve cell damage in the rat brain as defined by the optical microscope, Brit. J. exp. Path., 49 (1968) 87-106. 3 BRUIJN, W. C. DE, AND MCGEE-RUSSELL, S. M., Bridging a gap in pathology and histology, J. roy. micr. Soc., 85 0966) 77-90. 4 HAGER, H., HIRSCHBERGER, W., AND SCHOLZ, W., Electron microscopic changes in brain tissue of Syrian hamsters following acute hypoxia, Aerospace Med., 31 (1960) 379-387. 5 HAGER, H., Electron microscopical observations on the early changes in neurons caused by hypoxidosis and on the ultrastructural aspects of neuronal necrosis in the cerebral cortex of mammals. In J. P. SCHAD~ANDW. H. MCMENEMEY(Eds.), Selective Vulnerability of the Brain in Hypoxaemia, Blackwell, Oxford, 1963, pp. 125-136. 6 HILLS, C. P., The ultrastructure of anoxic-ischaemic lesions in the cerebral cortex of the adult rat brain, Guy's Hosp. Rep., 113 (1964) 333-348. 7 LUFT, J. H., Improvements in epoxy resin embedding methods, J. biophys, biochem. Cytol., 9 (1961) 409--414. 8 McGEE-RUSSELL,S. M., AND GOSZTONYI,G., Assembly of Semliki forest virus in brain, Nature (Lond.), 214 (1967) 1204-1206. 9 SCHOLZ,W., AND HAGER, H., Toxicity changes in the central nervous system. Oxygen deficiency and its influence on the central nervous system, U.S.A.F. Tech. (Final) Rep., Contract No. AF 61 (514)-945, Part II 0959) 1-24.
Brain Research, 20 (1970) 193-200
193
A COMBINED L I G H T AND ELECTRON MICROSCOPE STUDY OF EARLY ANOXIC-ISCHAEM1C CELL C H A N G E IN RAT BRAIN
S. M.
McGEE-RUSSELL*, A. W. BROWN ANDJ. B. BRIERLEY
Virus Research Unit and Neuropsychiatric Research Unit, Medical Research Council Laboratories, Carshalton, Surrey (Great Britain)
(Accepted November 21st, 1969)
INTRODUCTION Microvacuolation (MV) of the cytoplasm was described as the earliest neuronal alteration resulting from anoxia-ischaemia in the ipsilateral cerebral hemisphere of the rat 'Levine preparation'. It was demonstrable with the light microscope in conventionally stained paraffin sections of brains that had been fixed by perfusionl,L Microvacuolation was succeeded by ischaemic cell change (ICC), ischaemic cell change with incrustations and cell loss. Microvacuolation was recognizable after exposures to nitrogen of 30 and 20 min. In the present study a survival of 1 h after 40 rain exposure to nitrogen was chosen in order to observe transitional stages between MV and ICC. The ultrastructural examination was restricted to zone hl (CA1) of the hippocampus because of its high neuronal density and its known vulnerability to anoxiaischaemia. The purpose of the present investigation was to correlate the light microscopical appearances of microvacuolated neurones in thin large-area plastic sections 3,8 with those obtained with the electron microscope and to define their ultrastructural features, origin and associated subcellular changes. MATERIALSAND METHODS The experimental procedures have been describedl, 2. Four rats (White Wistar, Carshalton strain, weight 150 g) were exposed to nitrogen (5 litres/min) 0.25-3 h after application of the 'artery clasp' to the right common carotid artery. When apnoea occurred mechanical ventilation was employed until spontaneous respiration was resumed. The cycle, quiescence, hyperpnoea,
* Present address: Department of Biological Sciences, State University of New York at Albany, Albany, N.Y. 12203, U.S.A.
Brain Research, 20 (1970) 193-200
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s.M. MCGEE-RUSSELLet al.
convulsion, apnoea and mechanical ventilation, was repeated (average 10 times. range 4-17) during the 40 min. One hour later, under ether anaesthesia, the 'artery clasp' was removed and the brain was perfused with glutaraldehyde (6.5 ~ in cacodylate buffer, p H 7.4, minimum 250 ml during 20 rain) without initial perfusion with saline. The brain was left in situ for 2 h and was then removed and stored in the fixative at 4°C overnight. The forebrain was cut coronally through the pituitary stalk and a 1-2 m m slice posterior to this plane was embedded in plastic. The next posterior slice was embedded in Paraplast (Shandon Scientific Co. Ltd.) for light microscopical study. The EM slice was left in cacodylate buffer at 4°C for 24 h and post-fixed in osmium-cacodylate fixative (1% OsO4 in cacodylate buffer pH 7.4) at 4°C for 18 h. The slice was embedded in Epon 812 (5A:5B, LuftT). Sections were cut on a Porter Blum M T 1 ultra-microtome using sharpened steel edges to obtain sections 2-4/~m thick. These were stained with toluidine blue or cresyl fast violet at 60°C and were mounted in Epon on glass slides. Regions containing altered neurones in these sections were prepared as 'mesa pyramids' (1 mm × 1 mm) on the cut surfaces of the original blocks so that ultra-thin sections could be taken for electron microscopy. These were stained with 2 % uranyl acetate, lead citrate (animal IV only) and examined with an AEI EM 6 electron microscope at accelerating voltages of 60 and 40 kV, with 400 # m condenser apertures and 25/zm objective apertures. Paraffin sections (7 and t2 #m) were stained with cresyl fast violet, toluidine blue, Luxol fast blue and cresyl fast violet and with haematoxylin and eosin. The brains of 4 normal rats were fixed by perfusion with glutaraldehyde, removed 2-3 h later and all subsequent stages were carried out as above.
RESULTS
Controls
In the 4 animals the brain was uniformly tanned but appreciably shrunken. In paraffin and plastic sections (Fig. I A and B) staining was uniform and there was no evidence of artefact.
Fig. 1. A, Control rat. Hippocampus (hl). Paraffin section showing selective staining of neurones, endothelial cells and the absence of artefact. Cresyl fast violet, x 780. B, Control rat. Hippocampus (h!). Epon-embedded. Section (3/~m) showing general staining of all cellular elements and the absence of artifact. Cresyl fast violet, x 780. C, 'Levine preparation', ipsilateral hippocampus (hi). Animal IV. Paraffin section showing pyramidal cells containing microvacuoles separated by strongly basophilic cytoplasm. Toluidine blue. x 800. D, As for part C. Animal III. Epon-embedded. Section (3 #m) showing microvacuoles in neuronal cytoplasm and conspicuous perineuronal and perivascular spaces. Cresyl fast violet, x 800. E, As for part C. Animal III. 1 ~m section from 'mesa pyramid' showing microvacuoles evenly distributed and extending into the apical dendrite. Toluidine blue. x 1460. Brain Research, 20 (1970) 193-200
ANOXIC-ISCHAEMIC CELL CHANGE IN RAT
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Brain Research, 20 (1970) 193-200
196
S.M.
MCGEE-RUSSELL e[ ol.
Experimental animals Macroscopic In all 4 animals slight prominence of the inferior cerebellar vermis was evidence of minimal brain swelling. In coronal slices the ipsilateral neocortex was widened in all, least in animal i and most in animal lII.
Microscopic (a) Paraffin sections. Altered neurones were seen in the ipsilateral neocortex of all 4 animals but MV and ICC were seen in the hippocampus of only animals 1II and IV and in these two animals this structure was submitted to ultrastructural study. In the pyramidal cells of the hippocampus microvacuoles appeared as apparently empty circular spaces within a strongly basophilic cytoplasm (Fig. 1C). The nucleus was either normal or slightly shrunken, the nucleolus appeared normal and the cell was of normal size. (b) Plastic sections. Within the hippocampus (hl) there were no normal neurones. In a minority the cytoplasm showed a slight increase in staining intensity and contained a few microvacuoles; the nucleus and nucleolus were unaltered. The majority of neurones were of normal size and their darkly stained cytoplasm was virtually filled with microvacuoles (Fig. I D). A few microvacuoles could be seen in occasional intensely stained shrunken cells. Microvacuoles were usually circular but occasionally oval, they appeared to be empty and their diameters were 0.16-2.5 #m. They were evenly distributed in the cytoplasm and were often seen in the proximal portion of the apical dendrite (Fig. 1E). Increased cytoplasmic staining was associated with a reduced number of microvacuoles, shrinkage of the cell and a triangular darkly-staining nucleus.
Fig. 2. As for Fig. 1C. Animal IV. Low-power electron micrograph showing 2 neurones (n) containing microvacuoles (arrows). The cytoplasm is shrunken and the nucleus (centre) triangular. Swollen astrocytic processes (ap) surround the neurones and a blood vessel (bv). × 3375.
Brain Research, 20 (1970) 193-200
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Fig. 3. As for Fig. IC. Animal III. The microvacuoles (mv) are bounded by double membranes and some contain remnants of membranous material. Note the dilatation of the tubules and cisternae of the endoplasmic reticulum (arrows) and also occasional expansion of the nuclear membrane cisternae. There appears to be an accumulation of free ribosomes and ribosomal rosettes accompanied by an increase in the cytoplasmic matrix density. × 24,250.
Brain Research, 20 (1970) 193-200
198
s.M. MCGEE-RUSSELLel a/,
The conspicuous separation of the damaged neurones was due to wide perineuronal spaces, subdivided by trabeculae and either empty or containing a few cytoplasmic remnants. Similar spaces surrounded small blood vessels. Much smaller spaces gave a 'loosened' appearance to the adjacent neuropil. (e) Electron microscopy. Fig. 2 illustrates a typical microvacuolated cell. Each microvacuole consisted of an electron-lucent space bounded by two unit membranes (Figs. 3 and 4). Within the space there was often a small amount of high contrast material of ill-defined character probably representing remnants ofcristae. The majority of microvacuolated cells were surrounded by the swollen processes of astrocytes (Fig. 2) which corresponded to the perineuronal spaces seen with the light microscope. Microvacuolated cells were characterized by the absence of normal mitochondria but the presence of a double membrane around microvacuoles and the nature of their contents indicated their origin from mitochondria. Some dilatation of the tubules, vesicles and cisternae of the endoplasmic reticulum was also observed (Fig. 3). The material within the expanded membranes was of low electron density. There was an aggregation of free ribosomes and ribosomal rosettes together with an increased density of the cytoplasmic matrix (corresponding to the intensely basophilic microvacuolated neurones seen with the light microscope; Fig. IC-E).
Fig. 4. As for Fig. 1C. Animal III. High-power electron micrograph showing two swollen mitochondria containing remnants of cristae, x 68,300. Brain Research, 20 (1970) 193-200
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Within microvacuolated neurones, some nuclei were normal while others showed an aggregation of nuclear material sometimes accompanied by widening of the space between the nuclear membranes (Fig. 3). The majority of mitochondria in the swollen astrocytic processes and also in the adjacent neuropil appeared normal. DISCUSSION In the present investigation, the 'large area sectioning method' (refs. 3, 4) has facilitated the survey and detailed study of control and experimental brain tissue. Evidently the apparently empty microvacuoles seen in both paraffin and plastic sections are accounted for in most instances by swollen mitochondria and, in a minority, by dilatations within the endoplasmic reticulum. Hitherto, ultrastructural studies of the time course of anoxic-ischaemic alterations have been limited by either the employment of immersion-fixation6 or inadequate perfusion-fixation4,5,9. Hillse, using the Levine preparation, described swelling of astrocytic processes after 1.5 h but no alteration in neurone or capillary endothelium until 3 h. Hager et al. 4, Hager 5 and Scholz and Hager 9, using perfusion-fixation with osmic acid, studied the fine structure of the neocortex of the Syrian hamster killed immediately after repeated asphyxiation during a 12-15 min exposure to nitrogen. There was no parallel light microscopic study. Swelling of mitochondria was seen in neuronal cytoplasm of reduced rather than increased electron density. Such appearances are an artefact arising from inadequate perfusion-fixation, even in the brain of a normal animal (Brown and Brierley, unpublished observations). In the present study, the internal derangement of mitochondria implies some impairment of function of their oxidative enzymes. The parallel expansion of astrocytic processes implies that it has yet to be decided whether astrocytic or neuronal alterations are the first consequence of anoxia-ischaemia. SUMMARY Anoxic-ischaemic neuronal damage was studied by a combined light and electron microscope study of the brains of 4 rats exposed to nitrogen intermittently during a period of 40 min after interruption of blood flow in the right common carotid artery ('Levine preparation'). The animals were killed by perfusion-fixation with glutaraldehyde after survival of 1 h (measured from the end of exposure to nitrogen). Microvacuolation, the earliest alteration, was abundant in the ipsilateral hippocampi (hl) of two animals. The electron microscope study was restricted to this region. Microvacuoles in paraffin and plastic sections (light microscope) appeared as apparently empty circular spaces (0.16-2.5/zm in diameter) in the cytoplasm. At the ultrastructural level most microvacuoles were seen to be swollen mitochondria retaining their double membranes despite progressive disruption of their internal structure. Brain Research, 20 (1970) 193-200
s.M. MCGEE-RUSSELLet al.
200
Di l at at i o n s o f the tubules, vesicles and cisternae o f the e n d o p l a s m i c reticulum were also observed. T h e r e was an associated a p p a r e n t increase in r i b o so m es and in c y t o p l a s m i c m a t r i x density. The d a m a g e d n e u r o n e s were s u r r o u n d e d by swollen astrocytic processes.
REFERENCES 1 BROWN,A. W., AND BRIERLEY, J. B., Evidence for early anoxic-ischaemic cell damage in the rat brain, Experientia (Basel), 22 (1966) 546-547. 2 BROWN, A. W., AND BRIERLEY, J. B., The nature distribution and earliest stages of anoxic-ischaemic nerve cell damage in the rat brain as defined by the optical microscope, Brit. J. exp. Path., 49 (1968) 87-106. 3 BRUIJN, W. C. DE, AND MCGEE-RUSSELL, S. M., Bridging a gap in pathology and histology, J. roy. micr. Soc., 85 0966) 77-90. 4 HAGER, H., HIRSCHBERGER, W., AND SCHOLZ, W., Electron microscopic changes in brain tissue of Syrian hamsters following acute hypoxia, Aerospace Med., 31 (1960) 379-387. 5 HAGER, H., Electron microscopical observations on the early changes in neurons caused by hypoxidosis and on the ultrastructural aspects of neuronal necrosis in the cerebral cortex of mammals. In J. P. SCHAD~ANDW. H. MCMENEMEY(Eds.), Selective Vulnerability of the Brain in Hypoxaemia, Blackwell, Oxford, 1963, pp. 125-136. 6 HILLS, C. P., The ultrastructure of anoxic-ischaemic lesions in the cerebral cortex of the adult rat brain, Guy's Hosp. Rep., 113 (1964) 333-348. 7 LUFT, J. H., Improvements in epoxy resin embedding methods, J. biophys, biochem. Cytol., 9 (1961) 409--414. 8 McGEE-RUSSELL,S. M., AND GOSZTONYI,G., Assembly of Semliki forest virus in brain, Nature (Lond.), 214 (1967) 1204-1206. 9 SCHOLZ,W., AND HAGER, H., Toxicity changes in the central nervous system. Oxygen deficiency and its influence on the central nervous system, U.S.A.F. Tech. (Final) Rep., Contract No. AF 61 (514)-945, Part II 0959) 1-24.
Brain Research, 20 (1970) 193-200