Journal of the neurological Sciences, 1975, 26:235-244
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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The Effects of Glycerol on Cerebral Ultrastructure Following Experimentally Induced Cerebral Ischemia R. F. DODSON, Y. TAGASHIRA AND L. WAI-FONG CHU Departments of Neurology and Pathology, Baylor College of Medicine, and the Baylor-Methodist Centerfor Cerebrovascular Research, Houston, Texas 77025 (U.S.A.) (Received 26 February, 1975)
INTRODUCTION
The importance of limiting cerebral edema following a cerebrovascular accident or in disease conditions of the brain is a recognized necessity for improving the prognosis in these patients (Shaw, Alvord and Berry 1959; Plum 1964; Klatzo 1967; Ng and Nimmannitya 1970). The hyperosmolar agent, glycerol, has been shown to reduce greatly the deleterious effects associated with cerebral edema (Meyer, Charney, Rivera and Mathew 1971 ; Mathew, Meyer, Rivera, Charney and Hartmann 1972; Meyer, Fukuuchi, Shimazu, Ohuchi and Ericsson 1972a; Meyer, Teraura, Marx, Hashi and Sakamoto 1972b; Meyer, Shimazu, Ohuchi, Okamoto, Koto, Fukuuchi and Ericsson 1974; Ott, Mathew and Meyer 1974). It has been suggested that glycerol achieves its benefical effects through its direct action as a dehydrating agent, as well as through its direct and indirect contributions to metabolic improvement within edematous tissue. Morphological evaluation of the tissue response to glycerol therapy in cerebrovascular abnormalities was presented in a study involving experimentally-induced subarachnoid hemorrhage (Dodson, Hashi and Meyer 1973a) where glycerol was found to reduce effectively the degree of ultrastructural change associated with disturbed metabolism and edema (Hashi, Meyer, Shinmaru, Welch and Teraura 1972). The present investigation was designed to determine the effects of glycerol therapy on the tissue response in ischemic brain. The findings will be discussed with reference to changes reported in untreated ischemic tissue (Dodson, Kawamura, Aoyagi, Hartmann and Cheung 1973b; Dodson and Tagashira 1974). Specific sites of This work was supported by grant NS-09287 from the National Institute of Neurological Diseases and Stroke, and in part by grant RR-003350 from the General Clinical Research Centers Branch, Division of Research Resources, NIH, Bethesda, Md. 20014. Reprint request~ to: Ronald F. Dodson, Ph.D, Department ot Neurology, Baylor College of Medicine, 1200 Moursund Avenue, Houston, Texas 77025, U.S.A.
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UHU
apparent intraparenchymal preservation related to the glycerol-treated animals and a comparison with the intracellular changes noted in the untreated control group will be described. METHODS
The animals used in this experiment were adult squirrel monkeys divided into 5 groups based on respective periods of occlusive insult (½, 1, 2, 3, or 4 hr). Two animals from each period were maintained for a 3-day interval while 2 additional animals from each period were maintained for I week. The animals were lightly anesthetized (sodium pentobarbital) and a transorbital approach (Hudgins and Garcia 1970) was used to expose the right middle cerebral artery (RMCA) which was clipped for the selected period of either ½, 1. 2, 3, or 4 hr. The region of tissue involvement as well as the reproducibility of the model has been previously reported (Hudgins and Garcia 1970; Garcia and Kamijyo 1974) and were confirmed in the present experiment through light-microscopic studies. Blood pressure was monitored during the operative procedures as well as at selected intervals in the post-operative period. After the clip was removed, and RMCA flow re-established (Sundt, Grant and Garcia 1970) the animals were maintained under postoperative care for intervals up to 1 week. It has been shown previously that consistent but mild to moderate changes occurred after ½-2 hr of RMCA occlusion and advanced tissue alterations occurred in those groups subjected to 3 and 4 hr ofRMCA ischemia at 3 days post-occlusion, and at 1 week postocclusion (Dodson and Tagashira 1974). In the present study, animals were compared at post-occlusion intervals in an exactly comparable manner except that they received glycerol therapy. After the RMCA occlusion was performed the animals were immediately treated with a daily intravenous injection of 10~,~ glycerol in saline in a dosage o f0.08-0.8 g/kg body weight; this was injected rapidly over an interval of a few seconds in order to achieve comparable hyperosmolarity to that reported in man using a slow injection of 10~ glycerol over 40 min to 4 hr (Meyer et al. 1974). A second group of control animals was subjected to a sham operation, followed by similar daily injection of glycerol in comparable doses for a l-week period. After completing the two trials of therapy (either 3 days or 1 week) the animals were anesthetized with sodium pentobarbital and prepared for electron microscopy by intracardiac perfusion of 500 ml of 3 ~,~,glutaraldehyde in 0.1 M phosphate buffer. The brains were removed and coronal sections made at the level of the mammillary bodies, The tissue was inspected for homogeneity and completeness of fixation as judged by color and firmness, as well as vascular clearing of the major vessels. The tissue was also examined to assure that large areas of hemorrhage were not present, since the aim of the study was to study the effect of glycerol on ischemic infarction. Tissue samples were prepared from ischemic areas of insular, parietal, and temporal cortex, as well as from the corpus striatum and pallidum. Comparable areas from the contralateral nonischemic hemisphere were also taken. The tissues were post-stained in phosphate-buffered osmium tetroxide and processed in a standard manner for embedding in Spurr Embedding Media (Spurr 1969). Semi-thin plastic sections were prepared for light microscopy and selected areas were then sectioned for study with the RCA EMU-4 electron microscope.
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RESULTS
The animals were found to be normotensive during the operative procedures as well as in the postoperative state. Upon inspection of representative sections with a light microscope, the microvascular lumen were found to be free of debris. This observation confirms that flow had been re-established since the congestion associated with stasis would have prevented perfusate flow through the vascular bed. The findings were classified according to the time interval after RMCA occlusion, Control group No tissue changes were observed in the glycerol-treated sham-operated control group of animals at 1 week after exposure of, but without clipping the RMCA (Fig. 1). Glycerol-treated group in which RMCA was occluded for ½ hr The changes observed in the group after the RMCA was occluded for ½ hr and examined after sacrifice 1 week later consisted of a few scattered patches of minimal swelling within the perivascular elements of the astrocytes. These changes were limited to the right basal ganglia. Cortical areas showed no detectable abnormalities. Glycerol-treated ffroup, RMCA occluded for 1 hr Following 1 week of glycerol therapy, the changes noted were limited to the right putamen and globus pallidus. These changes were consistent with a minimal edema•tous response in that only the perivascular astrocytic foot processes were involved (Fig. 2). No edematous involvement was noted in the left hemisphere or in any other areas of the right hemisphere. Glycerol-treated group, RMCA occluded 2 hr No tissue alterations were observed in the left hemisphere (Fig. 3) but edematous involvement at 1 week post-occlusion was observed in tissue from the right side. Animals in this group showed limited ultrastructural changes in the form of intraceUular swelling of perivascular fibers of astrocytes within the globus pallidus, caudate nucleus and temporal cortex (Fig. 4). Increased numbers of lysosomal-like aggregates were present in the pericytes (Fig. 4). Glycerol-treated group, RMCA occluded 3 hr At 3 days post-operatively, the basal ganglia on the right side showed changes ranging from limited perivascular swelling in the putamen to more advanced swelling of both astrocytic foot processes and intraparenchymal astrocytic fibers in the globus pallidus and caudate nucleus (Fig. 5). Only focal areas of perivascular involvement were found in the right insular and temporal cortex. No changes were observed in the left hemisphere. Animals perfused after a 1-week period were noted to have less basal ganglia involvement than the above-mentioned 3-day group. Changes observed in the right globus pallidus consisted of slight swelling of foot processes of some astrocytes (Fig. 6). Lysosomes were present in astrocytic fibers and in pericytic cytoplasm (Fig. 6) Cortical areas on the right side appeared to be well-preserved with normal-appearing ultrastructure except for a few areas of slight perivascular astrocytic edema in the temporal regions.
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Fig. 1. Right insula; sham operated and glycerol: 7 days: components of interest include myelinated fibers (M), capillary (C), neuron (N), and neuropil fibers (n). x 5,440.
Fig. 2. Right putamen; 1hr occlusion and glycerol; 7 days: minor ultrastructural changes are limited to the immediate perivascular foot processes of the astrocytes (arrows). x 5,780.
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Fig. 3. Left globus pallidus; 2 hr occlusion and glycerol; 7 days; normal components include myelinated fibers (M), endothelial cell (E) of the capillary (C), mitochondria (m), and neuropil fibers (n). x 11,090.
Fig. 4. Right globus pallidus; 2 hr occlusion and glycerol; 7 days: tissue changes have been restricted to cytoplasmic swelling in the astrocytic fibers (A). Note that microfilaments (mf) and mitochondria (ml are structurally intact. Although the pericytic cytoplasm (P) is not swollen, "lysosomal-like" structures (arrows) are present, x 16,513.
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R . F . I)ODSON, Y. TAGASHIRA, k. Vv'AI-f.UNG CHt. r
Fig. 5. Right caudate: 3 hr occlusion and glycerol; 3 days: edematous changes within the perivasc~lar fiber (X) of the astrocytes also extends into astrocytic perikaryon (A). Although most mitochondria !m) within the cell body appear normal, some have associated minor ischemic changes (arrows). ~ (, 460
Fig. 6. Right caudate; 3 hr occlusion and glycerol: 7 days: the extent of edematous involvement ha~ become reduced compared with comparable zone of tissue illustrated in Fig. 5. Swollen areas are limited to the perivascular compartment (arrows) while "lysosomal-like'" structures (t) are found in the adjacent parenchyma and pericytes (P). × 5,780.
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Fig. 7. Right temporal cortex; 4 hr occlusion and glycerol; 3 days: typical perivascular edema within astrocytic fibers are shown (X). x 3,550.
Fig. 8. Right temporal cortex; 4 hr occlusion and glycerol ; 7 days: this ischemic area shows reduction of edematous involvement compared to the cortical region shown in Fig. 7. A neuron (N), perivascular oligodendroglia (O) as well as myelinated (M) and unmyelinated (UM) fibers are normal in appearance. × 4,500.
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Glycerol-treated group, R M C A occluded 4 hr
The group of monkeys studied after 3 days of daily glycerol injections showed extensive swelling of astrocytic foot processes and fibers within the basal ganglia. Likewise, cortical areas in the distribution of the RMCA showed a widespread swelling and cytoplasmic disorganization of the astrocytic fibers and perikaryon (Fig. 7). All regions of the left hemisphere were structurally intact. The group of animals perfused after daily injection of glycerol for 1 week showed less involvement in the basal ganglia and cortical areas in the RMCA distribution (Fig. 8) when compared to the 3-day group. Few lysosomal elements were noted and the extent of edematous involvement had receded to some degree so that only perivascular swelling of the astrocytic foot processes was present in the basal ganglia. Other areas of the right hemisphere as well as the contralateral regions showed no ultrastructural changes.
DISCUSSION
Acute changes in brain tissue which occur after temporary occlusion of the RMCA for ½, 1, 2, 3 or 4 hr intervals as used in the present experiments have been defined previously (Dodson et al. 1973b). Early post-ischemic changes in grey matter follow ultrastructural developments that may be classified into 3 stages. The first stage involves perivascular swelling of the astrocytic foot processes with an associated loss of glycogen stores within astrocytic cytoplasm. In the second stage the swelling extends into the astrocytic fibers and perikaryon, and in the third stage all cell types of ischemic parenchyma show swelling and disruption of ultrastructure. In a second set of experiments the ischemic responses of cerebral tissue to the time interval of temporary ischemia were compared after survival of the animals for 3 days and 1 week (Dodson and Tagashira 1974). When the present glycerol-treated groups were compared to the above-described untreated animals, the following differences were noted. Tissue alterations in the glycerol-treated animals were appreciably less than in ischemic regions of the comparable untreated groups o fanimals. Tissue swelling in the basal ganglion of the glyceroltreated animals was limited to the astrocytes. In the untreated animals with the RMCA occluded for 3 and 4 hr, changes were noted after 1 week which involved all cell types and included extracellular swelling. Furthermore, in the untreated animals, there was generally disintegration and degeneration of the cells of the basal ganglia at intervals of 3 days and 1 week post-occlusion. On the contrary, in the glycerol-treated groups, the basal ganglia at 1 week post-occlusion overall showed a preservation of structure and a decrease in edematous involvement. In other words, glycerol treatment resulted in preservation of organellular integrity. Ultrastructurally it appeared evident in all groups that the extent of edematous involvement was limited or reduced by glycerol therapy. Whether these effects are produced entirely by its hyperosmolar action or by means of some other metabolic function in the injured tissue, or by a combination of these effects, could not be defined by the present experiments. The edematous influence in the glycerol-treated tissues was limited to astrocytic components thus sparing involvement of the neuronal ele-
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ments, particularly the neurotransmitter-rich synaptasomal complexes. Another indicator of tissue stabilization which has been induced by glycerol therapy is shown by the lack of swelling within the pericytes as had been seen in the untreated groups. There were few if any ruptured membranes of the cytoplasmic organelles in the glycerol-treated group including mitochondria, endoplasmic reticulum, Golgi complexes and little dilution of cytoplasmic ground substance. All of these ultrastructural elements are essential for maintaining the metabolism and functions of the central nervous system. ACKNOWLEDGEMENTS
The authors would like to express their appreciation to Professor John Stirling Meyer for his helpful suggestions during the investigation and in the preparation of the manuscript. SUMMARY
Ultrastructural changes in cerebral tissue subjected to temporary occlusion on the right middle cerebral artery for intervals of 30 min, 1, 2, 3, and 4 hr were studied after daily injections of 109/o glycerol in saline. These changes were compared with previously reported data from untreated animals. The extent of tissue involvement was appreciably less in the glycerol-treated group as evidenced by the reduction of intracellular edema and fewer changes in the membranes of ultrastructural elements (mitochondria, endoplasmic reticulum, Golgi complex, and plasmalemma) known to be essential for cellular metabolism and function in the central nervous system. REFERENCES DODSON, R. F. AND Y. TAGASHIRA(! 974) Ultrastructural responses of cerebral tissue following periods of ischemic insult (presented at the 4th Annual Meeting of the Neuropathology Section of the Society for Neuroscience, St. Louis, Mo., 20-24 October). DODSON, R. F., K. HAsm AND J. S. MEYER (1973a) The effect of glycerol and intracarotid phenoxybenzamine after experimental subarachnoid hemorrhage - - An ultrastructural stu.dy, Acta neuropath. (Berl.), 24: 1-11. DODSON, R. F., Y. KAWAMURA,M. AOYA61, A. HARTMANUAND L. W. F. CHEUN6 (1973b) A comparative evaluation of the ultrastructural changes following induced cerebral infarction in the squirrel monkey and baboon, Cytobios, 8: 175-182. GARCIA, J. H. AND Y. KAMIJYO (1974) Cerebral infarction - - Evolution of histopathological changes after occlusion of a middle cerebral artery in primates, J. Neuropath. exp. Neurol., 33: 408--421. HASHI, K., J. S. MEYER. S. SHINMARU, K. M. A. WELCH AND T. TERAURA (1972) Effect of glycerol and intracarotid phenoxybenzamine on cerebral hemodynamics and metabolism after experimental subarachnoid hemorrhage, J. neurol. Sci., 17: 23-28. HUDGINS, W. R. AND J. H. GARClA (1970) Transorbital approach to the middle cerebral artery of the squirrel monkey - - A technique for experimental cerebral infarction applicable to ultrastructural studies, Stroke, 1: 107-111. KLATZO, I. (1967) Presidential address: Neuropathological aspects of brain edema, J. Neuropath. exp. Neurol., 26: 1-14. MATHEW, N. T., J. S. MEYER, V. M. RIVERA,J. Z. CHARNEYANDA. HARTMANN(1972) Double-blind evaluation of glycerol therapy in acute cerebral infarction, Lancet, 23 December, pp. 1327-1329. MEYER, J. S., J. Z. CHARNEY,V. i . RIVERAAND N. T. MATHEW(1971) Treatment with glycerol of cerebral edema due to acute cerebral infarction, Lancet, 6 November, pp. 993-997.
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MEYER, J. S., Y. FUKUUCH1, K. SHIMAZU, T. OHUCHI AND A. D. ERICSSON (1972a) Effect of intravenous infusion of glycerol on hemispheric blood flow and metabolism in patients with acute cerebral infarction. Stroke, 3: 168-180. MEYER, J. S., T. TERAURA,P. MARX, K. HASH1A~qDK. SAKAMOTO(1972b) Brain swelling due to experimental cerebral infarction - - Changes in vasomotor capacitance and effects of intravenous glycerol. Brain. 95 : 833-852. MEYER, J. S., K. SHIMAZU, T. OHUCH1, S. OKAMOTO, A. KOTO, Y. FUKUUCHI AND A. D. ERICSSON (1974) Cerebral metabolic effects of glycerol infusion in diabetics with stroke, J. neurol. Sci., 21 : 1-22. NG, L. K. Y. AND J. NIMMANNnOYA(1970) Massive cerebral infarction with brain swelling --- A clinicopathological study, Stroke, 1: 158-163. OTT, E. O., N. T. MATFmW AND J. S. MEYER (1974) Redistribution of regional cerebral blood flow after glycerol infus~ton in acute cerebral infarction, Neurology (Minneap.), 24:1117-1126. PLUM, F. (1964) Brain swelling and edema in cerebral vascular disease, Proc. Ass. Res. herr,, ment. Dis., 41: 318-348. SHAW, C. M., E. C. ALVORD, JR. AND R. G. BERRY (1959) Swelling the brain following ischemic infarction with arterial occlusion,Arch. Neurol. (Chic.), l: 161-177. SPURR, A. R. (1969) A low viscasity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res., 26: 3143. SUNDT, Z. M., W. G. GRANT AND J. H. GARClS (1970) Restoration of middle cerebral artery flow in experimental infarction, J. Neurosurg., 31: 311-322.
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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The Effects of Glycerol on Cerebral Ultrastructure Following Experimentally Induced Cerebral Ischemia R. F. DODSON, Y. TAGASHIRA AND L. WAI-FONG CHU Departments of Neurology and Pathology, Baylor College of Medicine, and the Baylor-Methodist Centerfor Cerebrovascular Research, Houston, Texas 77025 (U.S.A.) (Received 26 February, 1975)
INTRODUCTION
The importance of limiting cerebral edema following a cerebrovascular accident or in disease conditions of the brain is a recognized necessity for improving the prognosis in these patients (Shaw, Alvord and Berry 1959; Plum 1964; Klatzo 1967; Ng and Nimmannitya 1970). The hyperosmolar agent, glycerol, has been shown to reduce greatly the deleterious effects associated with cerebral edema (Meyer, Charney, Rivera and Mathew 1971 ; Mathew, Meyer, Rivera, Charney and Hartmann 1972; Meyer, Fukuuchi, Shimazu, Ohuchi and Ericsson 1972a; Meyer, Teraura, Marx, Hashi and Sakamoto 1972b; Meyer, Shimazu, Ohuchi, Okamoto, Koto, Fukuuchi and Ericsson 1974; Ott, Mathew and Meyer 1974). It has been suggested that glycerol achieves its benefical effects through its direct action as a dehydrating agent, as well as through its direct and indirect contributions to metabolic improvement within edematous tissue. Morphological evaluation of the tissue response to glycerol therapy in cerebrovascular abnormalities was presented in a study involving experimentally-induced subarachnoid hemorrhage (Dodson, Hashi and Meyer 1973a) where glycerol was found to reduce effectively the degree of ultrastructural change associated with disturbed metabolism and edema (Hashi, Meyer, Shinmaru, Welch and Teraura 1972). The present investigation was designed to determine the effects of glycerol therapy on the tissue response in ischemic brain. The findings will be discussed with reference to changes reported in untreated ischemic tissue (Dodson, Kawamura, Aoyagi, Hartmann and Cheung 1973b; Dodson and Tagashira 1974). Specific sites of This work was supported by grant NS-09287 from the National Institute of Neurological Diseases and Stroke, and in part by grant RR-003350 from the General Clinical Research Centers Branch, Division of Research Resources, NIH, Bethesda, Md. 20014. Reprint request~ to: Ronald F. Dodson, Ph.D, Department ot Neurology, Baylor College of Medicine, 1200 Moursund Avenue, Houston, Texas 77025, U.S.A.
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UHU
apparent intraparenchymal preservation related to the glycerol-treated animals and a comparison with the intracellular changes noted in the untreated control group will be described. METHODS
The animals used in this experiment were adult squirrel monkeys divided into 5 groups based on respective periods of occlusive insult (½, 1, 2, 3, or 4 hr). Two animals from each period were maintained for a 3-day interval while 2 additional animals from each period were maintained for I week. The animals were lightly anesthetized (sodium pentobarbital) and a transorbital approach (Hudgins and Garcia 1970) was used to expose the right middle cerebral artery (RMCA) which was clipped for the selected period of either ½, 1. 2, 3, or 4 hr. The region of tissue involvement as well as the reproducibility of the model has been previously reported (Hudgins and Garcia 1970; Garcia and Kamijyo 1974) and were confirmed in the present experiment through light-microscopic studies. Blood pressure was monitored during the operative procedures as well as at selected intervals in the post-operative period. After the clip was removed, and RMCA flow re-established (Sundt, Grant and Garcia 1970) the animals were maintained under postoperative care for intervals up to 1 week. It has been shown previously that consistent but mild to moderate changes occurred after ½-2 hr of RMCA occlusion and advanced tissue alterations occurred in those groups subjected to 3 and 4 hr ofRMCA ischemia at 3 days post-occlusion, and at 1 week postocclusion (Dodson and Tagashira 1974). In the present study, animals were compared at post-occlusion intervals in an exactly comparable manner except that they received glycerol therapy. After the RMCA occlusion was performed the animals were immediately treated with a daily intravenous injection of 10~,~ glycerol in saline in a dosage o f0.08-0.8 g/kg body weight; this was injected rapidly over an interval of a few seconds in order to achieve comparable hyperosmolarity to that reported in man using a slow injection of 10~ glycerol over 40 min to 4 hr (Meyer et al. 1974). A second group of control animals was subjected to a sham operation, followed by similar daily injection of glycerol in comparable doses for a l-week period. After completing the two trials of therapy (either 3 days or 1 week) the animals were anesthetized with sodium pentobarbital and prepared for electron microscopy by intracardiac perfusion of 500 ml of 3 ~,~,glutaraldehyde in 0.1 M phosphate buffer. The brains were removed and coronal sections made at the level of the mammillary bodies, The tissue was inspected for homogeneity and completeness of fixation as judged by color and firmness, as well as vascular clearing of the major vessels. The tissue was also examined to assure that large areas of hemorrhage were not present, since the aim of the study was to study the effect of glycerol on ischemic infarction. Tissue samples were prepared from ischemic areas of insular, parietal, and temporal cortex, as well as from the corpus striatum and pallidum. Comparable areas from the contralateral nonischemic hemisphere were also taken. The tissues were post-stained in phosphate-buffered osmium tetroxide and processed in a standard manner for embedding in Spurr Embedding Media (Spurr 1969). Semi-thin plastic sections were prepared for light microscopy and selected areas were then sectioned for study with the RCA EMU-4 electron microscope.
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RESULTS
The animals were found to be normotensive during the operative procedures as well as in the postoperative state. Upon inspection of representative sections with a light microscope, the microvascular lumen were found to be free of debris. This observation confirms that flow had been re-established since the congestion associated with stasis would have prevented perfusate flow through the vascular bed. The findings were classified according to the time interval after RMCA occlusion, Control group No tissue changes were observed in the glycerol-treated sham-operated control group of animals at 1 week after exposure of, but without clipping the RMCA (Fig. 1). Glycerol-treated group in which RMCA was occluded for ½ hr The changes observed in the group after the RMCA was occluded for ½ hr and examined after sacrifice 1 week later consisted of a few scattered patches of minimal swelling within the perivascular elements of the astrocytes. These changes were limited to the right basal ganglia. Cortical areas showed no detectable abnormalities. Glycerol-treated ffroup, RMCA occluded for 1 hr Following 1 week of glycerol therapy, the changes noted were limited to the right putamen and globus pallidus. These changes were consistent with a minimal edema•tous response in that only the perivascular astrocytic foot processes were involved (Fig. 2). No edematous involvement was noted in the left hemisphere or in any other areas of the right hemisphere. Glycerol-treated group, RMCA occluded 2 hr No tissue alterations were observed in the left hemisphere (Fig. 3) but edematous involvement at 1 week post-occlusion was observed in tissue from the right side. Animals in this group showed limited ultrastructural changes in the form of intraceUular swelling of perivascular fibers of astrocytes within the globus pallidus, caudate nucleus and temporal cortex (Fig. 4). Increased numbers of lysosomal-like aggregates were present in the pericytes (Fig. 4). Glycerol-treated group, RMCA occluded 3 hr At 3 days post-operatively, the basal ganglia on the right side showed changes ranging from limited perivascular swelling in the putamen to more advanced swelling of both astrocytic foot processes and intraparenchymal astrocytic fibers in the globus pallidus and caudate nucleus (Fig. 5). Only focal areas of perivascular involvement were found in the right insular and temporal cortex. No changes were observed in the left hemisphere. Animals perfused after a 1-week period were noted to have less basal ganglia involvement than the above-mentioned 3-day group. Changes observed in the right globus pallidus consisted of slight swelling of foot processes of some astrocytes (Fig. 6). Lysosomes were present in astrocytic fibers and in pericytic cytoplasm (Fig. 6) Cortical areas on the right side appeared to be well-preserved with normal-appearing ultrastructure except for a few areas of slight perivascular astrocytic edema in the temporal regions.
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Fig. 1. Right insula; sham operated and glycerol: 7 days: components of interest include myelinated fibers (M), capillary (C), neuron (N), and neuropil fibers (n). x 5,440.
Fig. 2. Right putamen; 1hr occlusion and glycerol; 7 days: minor ultrastructural changes are limited to the immediate perivascular foot processes of the astrocytes (arrows). x 5,780.
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Fig. 3. Left globus pallidus; 2 hr occlusion and glycerol; 7 days; normal components include myelinated fibers (M), endothelial cell (E) of the capillary (C), mitochondria (m), and neuropil fibers (n). x 11,090.
Fig. 4. Right globus pallidus; 2 hr occlusion and glycerol; 7 days: tissue changes have been restricted to cytoplasmic swelling in the astrocytic fibers (A). Note that microfilaments (mf) and mitochondria (ml are structurally intact. Although the pericytic cytoplasm (P) is not swollen, "lysosomal-like" structures (arrows) are present, x 16,513.
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Fig. 5. Right caudate: 3 hr occlusion and glycerol; 3 days: edematous changes within the perivasc~lar fiber (X) of the astrocytes also extends into astrocytic perikaryon (A). Although most mitochondria !m) within the cell body appear normal, some have associated minor ischemic changes (arrows). ~ (, 460
Fig. 6. Right caudate; 3 hr occlusion and glycerol: 7 days: the extent of edematous involvement ha~ become reduced compared with comparable zone of tissue illustrated in Fig. 5. Swollen areas are limited to the perivascular compartment (arrows) while "lysosomal-like'" structures (t) are found in the adjacent parenchyma and pericytes (P). × 5,780.
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Fig. 7. Right temporal cortex; 4 hr occlusion and glycerol; 3 days: typical perivascular edema within astrocytic fibers are shown (X). x 3,550.
Fig. 8. Right temporal cortex; 4 hr occlusion and glycerol ; 7 days: this ischemic area shows reduction of edematous involvement compared to the cortical region shown in Fig. 7. A neuron (N), perivascular oligodendroglia (O) as well as myelinated (M) and unmyelinated (UM) fibers are normal in appearance. × 4,500.
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Glycerol-treated group, R M C A occluded 4 hr
The group of monkeys studied after 3 days of daily glycerol injections showed extensive swelling of astrocytic foot processes and fibers within the basal ganglia. Likewise, cortical areas in the distribution of the RMCA showed a widespread swelling and cytoplasmic disorganization of the astrocytic fibers and perikaryon (Fig. 7). All regions of the left hemisphere were structurally intact. The group of animals perfused after daily injection of glycerol for 1 week showed less involvement in the basal ganglia and cortical areas in the RMCA distribution (Fig. 8) when compared to the 3-day group. Few lysosomal elements were noted and the extent of edematous involvement had receded to some degree so that only perivascular swelling of the astrocytic foot processes was present in the basal ganglia. Other areas of the right hemisphere as well as the contralateral regions showed no ultrastructural changes.
DISCUSSION
Acute changes in brain tissue which occur after temporary occlusion of the RMCA for ½, 1, 2, 3 or 4 hr intervals as used in the present experiments have been defined previously (Dodson et al. 1973b). Early post-ischemic changes in grey matter follow ultrastructural developments that may be classified into 3 stages. The first stage involves perivascular swelling of the astrocytic foot processes with an associated loss of glycogen stores within astrocytic cytoplasm. In the second stage the swelling extends into the astrocytic fibers and perikaryon, and in the third stage all cell types of ischemic parenchyma show swelling and disruption of ultrastructure. In a second set of experiments the ischemic responses of cerebral tissue to the time interval of temporary ischemia were compared after survival of the animals for 3 days and 1 week (Dodson and Tagashira 1974). When the present glycerol-treated groups were compared to the above-described untreated animals, the following differences were noted. Tissue alterations in the glycerol-treated animals were appreciably less than in ischemic regions of the comparable untreated groups o fanimals. Tissue swelling in the basal ganglion of the glyceroltreated animals was limited to the astrocytes. In the untreated animals with the RMCA occluded for 3 and 4 hr, changes were noted after 1 week which involved all cell types and included extracellular swelling. Furthermore, in the untreated animals, there was generally disintegration and degeneration of the cells of the basal ganglia at intervals of 3 days and 1 week post-occlusion. On the contrary, in the glycerol-treated groups, the basal ganglia at 1 week post-occlusion overall showed a preservation of structure and a decrease in edematous involvement. In other words, glycerol treatment resulted in preservation of organellular integrity. Ultrastructurally it appeared evident in all groups that the extent of edematous involvement was limited or reduced by glycerol therapy. Whether these effects are produced entirely by its hyperosmolar action or by means of some other metabolic function in the injured tissue, or by a combination of these effects, could not be defined by the present experiments. The edematous influence in the glycerol-treated tissues was limited to astrocytic components thus sparing involvement of the neuronal ele-
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ments, particularly the neurotransmitter-rich synaptasomal complexes. Another indicator of tissue stabilization which has been induced by glycerol therapy is shown by the lack of swelling within the pericytes as had been seen in the untreated groups. There were few if any ruptured membranes of the cytoplasmic organelles in the glycerol-treated group including mitochondria, endoplasmic reticulum, Golgi complexes and little dilution of cytoplasmic ground substance. All of these ultrastructural elements are essential for maintaining the metabolism and functions of the central nervous system. ACKNOWLEDGEMENTS
The authors would like to express their appreciation to Professor John Stirling Meyer for his helpful suggestions during the investigation and in the preparation of the manuscript. SUMMARY
Ultrastructural changes in cerebral tissue subjected to temporary occlusion on the right middle cerebral artery for intervals of 30 min, 1, 2, 3, and 4 hr were studied after daily injections of 109/o glycerol in saline. These changes were compared with previously reported data from untreated animals. The extent of tissue involvement was appreciably less in the glycerol-treated group as evidenced by the reduction of intracellular edema and fewer changes in the membranes of ultrastructural elements (mitochondria, endoplasmic reticulum, Golgi complex, and plasmalemma) known to be essential for cellular metabolism and function in the central nervous system. REFERENCES DODSON, R. F. AND Y. TAGASHIRA(! 974) Ultrastructural responses of cerebral tissue following periods of ischemic insult (presented at the 4th Annual Meeting of the Neuropathology Section of the Society for Neuroscience, St. Louis, Mo., 20-24 October). DODSON, R. F., K. HAsm AND J. S. MEYER (1973a) The effect of glycerol and intracarotid phenoxybenzamine after experimental subarachnoid hemorrhage - - An ultrastructural stu.dy, Acta neuropath. (Berl.), 24: 1-11. DODSON, R. F., Y. KAWAMURA,M. AOYA61, A. HARTMANUAND L. W. F. CHEUN6 (1973b) A comparative evaluation of the ultrastructural changes following induced cerebral infarction in the squirrel monkey and baboon, Cytobios, 8: 175-182. GARCIA, J. H. AND Y. KAMIJYO (1974) Cerebral infarction - - Evolution of histopathological changes after occlusion of a middle cerebral artery in primates, J. Neuropath. exp. Neurol., 33: 408--421. HASHI, K., J. S. MEYER. S. SHINMARU, K. M. A. WELCH AND T. TERAURA (1972) Effect of glycerol and intracarotid phenoxybenzamine on cerebral hemodynamics and metabolism after experimental subarachnoid hemorrhage, J. neurol. Sci., 17: 23-28. HUDGINS, W. R. AND J. H. GARClA (1970) Transorbital approach to the middle cerebral artery of the squirrel monkey - - A technique for experimental cerebral infarction applicable to ultrastructural studies, Stroke, 1: 107-111. KLATZO, I. (1967) Presidential address: Neuropathological aspects of brain edema, J. Neuropath. exp. Neurol., 26: 1-14. MATHEW, N. T., J. S. MEYER, V. M. RIVERA,J. Z. CHARNEYANDA. HARTMANN(1972) Double-blind evaluation of glycerol therapy in acute cerebral infarction, Lancet, 23 December, pp. 1327-1329. MEYER, J. S., J. Z. CHARNEY,V. i . RIVERAAND N. T. MATHEW(1971) Treatment with glycerol of cerebral edema due to acute cerebral infarction, Lancet, 6 November, pp. 993-997.
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MEYER, J. S., Y. FUKUUCH1, K. SHIMAZU, T. OHUCHI AND A. D. ERICSSON (1972a) Effect of intravenous infusion of glycerol on hemispheric blood flow and metabolism in patients with acute cerebral infarction. Stroke, 3: 168-180. MEYER, J. S., T. TERAURA,P. MARX, K. HASH1A~qDK. SAKAMOTO(1972b) Brain swelling due to experimental cerebral infarction - - Changes in vasomotor capacitance and effects of intravenous glycerol. Brain. 95 : 833-852. MEYER, J. S., K. SHIMAZU, T. OHUCH1, S. OKAMOTO, A. KOTO, Y. FUKUUCHI AND A. D. ERICSSON (1974) Cerebral metabolic effects of glycerol infusion in diabetics with stroke, J. neurol. Sci., 21 : 1-22. NG, L. K. Y. AND J. NIMMANNnOYA(1970) Massive cerebral infarction with brain swelling --- A clinicopathological study, Stroke, 1: 158-163. OTT, E. O., N. T. MATFmW AND J. S. MEYER (1974) Redistribution of regional cerebral blood flow after glycerol infus~ton in acute cerebral infarction, Neurology (Minneap.), 24:1117-1126. PLUM, F. (1964) Brain swelling and edema in cerebral vascular disease, Proc. Ass. Res. herr,, ment. Dis., 41: 318-348. SHAW, C. M., E. C. ALVORD, JR. AND R. G. BERRY (1959) Swelling the brain following ischemic infarction with arterial occlusion,Arch. Neurol. (Chic.), l: 161-177. SPURR, A. R. (1969) A low viscasity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res., 26: 3143. SUNDT, Z. M., W. G. GRANT AND J. H. GARClS (1970) Restoration of middle cerebral artery flow in experimental infarction, J. Neurosurg., 31: 311-322.
