Morphology of tissue damage due to experimental cerebral ischemia in rats

Exp. Pathol. 1988; 35: 219-230 VEB Gustav Fischer Verlag lena

Department of Neuropathology and Institute of Phannacology and Toxicology, Philipps University Marburg (Lahn), F.R.G.

Morphology of tissue damage due to experimental cerebral ischemia in rats By H. D. MENNEL, D. SAUER, C. ROSSBERG, G. W. BIELENBERG and J. KRIEGLSTEIN With 5 figures Address for correspondence: Prof. Dr. H. D. MENNEL, Abteilung Neuropathologie, Zentrum Pathologie, Universitat Marburg, Klinikum Lahnberge, D - 3550 Marburg (Lahn), F.R.G. Key words: cerebral ischemia; ischemic cell damage; experimental brain infarction

Summary

Two models of experimental cerebral ischemia in rats were developed and used. The first model was permanent occlusion of both carotids up to 3 weeks, the second model the temporal occlusion of both carotids and systemic hypotension for 10 min. Rats treated by the first experimental set were investigated after one, 2 and 3 weeks. In all groups, about 40 % of so treated animals had territorial infarcts, often more than one in the animal in question. These infarcts developed from necrotic, pale areas to ischemic cysts and this copied the evolution of human territorial infarction. Astroglial reaction was only seen in the border zone. In the second model, rats preferentially developed, as known, the so called delayed ganglion cell necrosis in the field CA 1 of the hippocampus. Cells were not altered on the second, but damaged on the sixth day after experimental ischemia. In both models the hippocampus was damaged, however in the first the damages were morphologically distinct from the damage in carotid occlusion in systemic hypotension. The first experimental model suits better for human territorial infarction, the second is highly reproducible and thus provides a much better experimental tool. 1. Introduction Cerebral ischemia in experimental animals has long been used to investigate the pathogenesis of ischemic damage and its therapy. COLMANT (1965) performed unilateral carotid occlusion combined with hypoxia to induce neuronal changes in one hemisphere of the brain. Lately, several models of ischemia in rats (PULSINELLI et al. 1979, SMITH et al. 1984) and gerbils (KIRINO and SANO 1984) with high reproducibility were developed to study different types of ischemic cell injury in the brain. The most striking finding was the occurrence of delayed neuronal damage in the field CA 1 of ammon's horn distinct from the classical ischemic cell alteration. The mechanism of this specific "delayed" event is not completely understood. In the human, several morphological forms of ischemia including elective necrosis in zones of "selective vulnerability" (SPIELMEYER 1928) in the hippocampus can be observed. In addition, other and more common types of ischemic conditions occur in man, especially complete liquefied necroses of vascular territories of selective neuronal death in cortical areas or basal ganglia, hemorrhagic infarction or rarely permanent coagulation necrosis. These variegated features of ischemic sequelae in the brain together with changing localisation and differing pathogenesis make it difficult for the pathologist and clinician to look at cerebral ischemia as a uniform phenomenon. Exp. Pathol. 35 (1988) 4

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Thus, also models of experimental ischemia have to be carefully analyzed as to the type of ischemic damage they represent. We made use of 2 different arrays of experimental ischemia leading to largely differing results in order to better understand the sequence of events in ischemic conditions from the morphological point of view. These 2 models were: First: A model of bilateral carotid occlusion and systemic hypotension (SMITH et al. 1984) and Second: The permanent ligature of both carotids for 1 to 3 weeks. The aim of this paper is to mainly summarize the results of the second model, since in this experimental set a wide range of different morphological states and features of ischemic damage was observed. We intend to infer this simple model as suited for the study of a pattern of morphological findings in cerebro-vascular insufficiency. Yet, the share of affected animals is too low to permit reproducible therapeutic studies. The first model will be mentioned only briefly and in some aspects, since the results share some common features with those obtained in permanent occlusion. The first model seems highly reproducible, therefore suited for analysis of therapy and prevention of damage, but obviously does not or only partly represent the most common type of "stroke" in man. More comprehensive results of a whole pattern of such experiments including biochemical and other aspects of this model were given elsewhere (BIELENBERG et al. 1987). 2. Materials and Methods Short term carotid occlusion A sample of 16 rats of Wistar strain was held without food for one day. Both carotids were then occluded by clips and the blood pressure lowered to about 40 mm Hg by exsanguination and infusion oftrimethaphan (Arfonade®). Body temperature and EEG were continuously recorded; the rats were artificially ventilated. After 10 min the clips were removed from both carotids and blood pressure raised to normal values by reinfusion of the shedded blood. The rats were allowed to recover and survived for 2,5, 7 and 14 d. The same procedures were performed with rats of Fisher inbred strain. These rats were much more susceptible to ischemia and did not survive the experiments. Thus, a slightly modified experimental set in Fisher rats was used: They were subjected to 8 or 9 min of ischemia and lowering of blood pressure to 50 mm Hg. From 13 animals treated in this way only 6 survived for 7 d. Permanent ligature 49 rats were treated as follows: Both carotids were occluded permanently without reopening during the remaining lifetime of the animals. The rats were separated into 3 groups with survival times of 7, 14 and 20 d, respectively. 3 of the rats of the same strain were treated by unilateral permanent carotid occlusion. All rats were perfused with buffered formalin solution (4 %/pH 7.4) after the respective survival times. The brains were then removed and the fixation in buffered formalin was continued for some days. Coronal sections were then performed depending on the experiment in question. All brains of rats with permanent carotid occlusion were cut in serial sections. Brains of animals with short term occlusion and hypotension of another series had been previously cut in serial sections in order to analyse the alterations in all cerebral regions (SAUER 1987). After it became clear that the main region of damage was invariably to be found in a plane 6 mm frontal to the tentorium, this plane only was looked at in all following experiments including those reported in this paper. This corresponds to the plane - 3.3 from bregma and 5.8 from interauricular. From this "reference plane", some serial sections were performed and taken for the different staining methods. Brains or part of the brains were embedded in paraffin and cut in 6 micron thick slices. The following staining procedures were used: cresylviolet staining for neurons, celestin-blue-acidfuchsin staining for the demonstration of ischemic cell damage, Kanzler's stain or PTAH in order to visualize astroglial cells, Bodian's silver impregnation to give evidence ofaxons and the Heidenhain-Woe1cke impregnation for myelin. Immunohistological methods were used in addition to follow the astroglial reaction to the damage by demonstration of the glial fibrillary acid protein using the PAP-method. 220

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3. Results 3.1. Quanti tati ve res ults 3.1.1. Permanent carotid occlusion (table 1) 3.1.1.1. Group 1: Seven days survival: Out of 17 treated rats, 5 developed substantial lesions in different regions of the brain. On the whole, 11 lesions have been observed in the cerebellum, basal ganglia, ammon's horn and cortex. No damage was observed in the brainstem proper. Table 1. Number of substantial brain lesions in rats treated by permanent carotid occlusion

Number of treated rats Number of affected rats Number of substantial lesions Localisation Ammon's horn Basal ganglia Cortex Midbrain Cerebellum Number of lesions per rat

1 week duration

2 weeks duration

3 weeks duration

Total

17

17

5

5

11

13

15 6 14

49 16 38

4 4 3

13 12 6 2 6

2 4 2 1 2 2.2

7 4

2.6

3 2.3

3.1.1.2. Group 2: Fourteen days survival: In this group of 17 rats, 5 had pathologic findings localized in the cerebral cortex, striatum, cerebellar cortex, ammon's horn and pons. In these 5 rats, 13 independent hypoxic regions were encountered. Cellular necroses in ammon's horn were usually restricted to the CA 1 field. However, in one instance, a circumscript damage in CA4 was in addition noted. The exceptional necrosis in the brain stem occurred in this group. 3. 1. 1.3. Group 3: Twenty one days survival: 6 animals with 14 circumscribed hypoxic lesions were met in this group. There were on the whole, 3 infarcts in cortical areas of cerebrum and cerebellum each, and 2 in the striatum and thalamus, respectively. 4 areas of ischemic pyramidal cell alterations were found in ammon's horn, one of them in the unique localisation in CA3 exclusively, the others as usual, in CA 1. A survey of the statistical results is given in table 1. 3.1.2. Temporary carotid occlusion and hypotension 3.1.2.1. Group 1: Two days survival: 3 rats, which had been treated by combined occlusion and hypotension for 10 min, fell into this group: They survived only 2 d; at this time, changes in ammon's horn were almost completely absent. In only one rat, 0.4 % of pyramidal cells in the layer CA 1 unilaterally were visibly damaged. 3.1.2.2. Group 2: Five days survival: 3 rats which belonged in this group had visible ischemic necroses in the layer CA 1 in ammon's horn. The percentage of shrunken cells ranged from 0 % (one animal unilaterally) to 75% (one rat unilaterally), the arithmetic mean being 28%, taken from 6 independently evaluated layers CA 1 in the 3 rats. 3.1.2.3. Group 3: Seven days survival: In this group of 3 animals the share of hypoxic cells in the CA 1 region of ammon's horn was between 69 and 94 % of all pyramids of this layer. The arithmetic Exp. Pathol. 35 (1988) 4

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mean was as high as 87 %. At this time, reactive gliosis was noted to a considerable extent; however, no attempt to quantify this reaction was undertaken. 3.1.2.4. Group 4: In this group of again 3 animals the average of damaged cells in the 6 independently counted regions CA 1 of ammon's hom was 53 %, ranging from 0 to 79. 3.2. Morphological results 3.2.1. Permanent occlusion 3.2.1.1. Group 1: One week survival: The features of the alterations in ammon's hom were correspondent to the one found in occlusion and systemic hypotension; in other areas - cerebellum, basal ganglia and cortex - the lesions were identical or very similar to each other. The main patterns in the latter localisations were reduction of cells, weak stainability for anilin dyes (Erbleichung) and sponginess (fig. I a, b). The preservation of shrunken ischemic neurons at this time was exceptional (fig. 1 c). In one instance, the beginning gliosis within the zone of pallor was noted (fig. 1d). 3.2.1.2. Group 2: Two weeks survival: In ammon's hom as in carotid occlusion with hypotension after this time many ischemic pyramidal cells were visible. In other areas, the main features of infarcted areas were increased cellularity up to the formation of fat granule cells as well as occurrence oflarger cysts (fig. 2a, b). Cystic transformation of the whole field with abundance of fat granule cells however was the exception only met once (fig.2c). The common type of lesion depicted pronounced status spongiosus in combination with densely packed (microglial) cells, some of which were already transformed into lipid phagocytes (fig. 2d). 3.2.1.3. Group 3: Three weeks survival: In this group, the greatest variation of findings had been noted; first, there were 2 kinds of damage in the hippocampus. In the field CA 1, shrunken neurons in affected area were yet present. From both sides, the virtual subventricular as well as the virtual sub arachnoidal , rod shaped microglial cells and astrocytes were met with. Astrocytes depicted nicely with the GFAPmethod, accumulated in high number within affected areas (fig. 3a, b). There

Fig. I a. Pallor (Erbleichungszone) in the cortical area: circumscribed region of decreased affinity to anilin dyes, due to lack of cell nuclei and structures of the neuropil. Top of the picture: sub arachnoidal space; bottom: ammon's hom. Cresylviolet, X 50. Fig. 1 b. Region in the cerebellum: The layer of Purkinje cells shows complete absence or ischemic change of perikarya. In the stratum moleculare one finds in addition to diminished affinity to anilin dyes a prominent status spongiosus. Cresylviolet, X 125. Fig. 1 c. Remaining neurons in the border zone of a focus of pallor. Ischemic cell change in advanced stage: shrinking and hyperchromasia. Cresylviolet, X 200. Fig. 1 d. Starting cellular infiltration in pallor zone within the corpus striatum. Cresylviolet, X 50. Fig. 2a. In the center of necrosis there is a, dense infiltration of rod shaped and round cells. The surrounding tissue shows pronounced pallor and status spongiosus. There is not yet cystic transformation of the tissue. Cresylviolet, X 50. Fig. 2 b. In higher magnification one sees that in addition to wideley open vessels there is increasing sponginess of the tissue by accumulation of small, confluent cystic holes. Cresylviolet, X 200. Fig. 2c. Cellular infiltration during advanced cystic transformation consists of fat granule cells which are loosly arranged. Furthermore one finds capillaries. Cresylviolet, X 125. Fig. 2d. Mixture of advanced cystic transformation and cellular infiltration of the fat granule cell type. Cresylviolet, X 125. 222

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Fig. 1.

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Fig . 2.

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was a slightly altered picture in the region CA3, which was exceptionally damaged in one instance in this group. Here too, a dense network of astrocytes with abundant processes had been formed; yet in contrast to the findings in CA 1, no neurons could be identified (fig. 3 c, d). The lesions outside the hippocampus had 2 features depending on the present or absent formation of larger cysts: In this latter case, there was an inner ring around the cyst formed by fat granule cells and an outer ring, which consisted entirely of astrocytes. The demonstration of astrocytic processes was especially clear in liquefied necroses of the cerebellum, where this population is obviously represented by the Bergmann glia (fig. 4a). Smaller necroses, not yet completely liquefied, had in their center a dense accumulation of fat granule cells and around a prominent rim of astrocytes (fig. 4b). The damaged tissue in this stage had a pronounced tendency to show calcification. Lastly, there were few other fields, especially in the basal ganglia, where the neurons were partly preserved, no fat granule cells were seen, but a diffuse increase in astrocytes as well as calcification occurred. 3.2.2. Temporary occlusion and hypotension All groups in this experimental array had only major pathological findings in the field CA I of the hippocampus. Minor damage in some cortical areas, basal ganglia and field CA4 of ammon's hom will not be considered here. The findings in the groups with 2,5, 7 and 14 days of survival show a common tendency; the findings of the 3 groups with permanent occlusion -if only field CA 1 of ammon's hom is considered - fit equally well into this sequence of alterations. Beginning with day 5, shrunken, pale pyramidal cells became obvious. Nuclei were homogeneous and elongated. The cells and nuclei stained dark with cresyl violett and red with acid fuchsin. The highest relative number of those neurons was found after 1 week. Yet they could equally be observed after three weeks, when in addition a homogenizing of the whole cell had taken place (fig. 5a). By morphometry only it became clear, that after 2 weeks time, a relative loss of neurons had begun. Our experiments do not show, how fast or slow this process went on. From the day 5 onward, both rod like (micro-) glial and astroglial cells started to proliferate. Astrocytes were seen first in the direct neighbourhood of the vessels (fig. 5b), which mark the prolongation of the subarachnoidal space (sulcus hippocampi). Second, astrocytes were seen near the virtual or manifest ventricular ependyma, but as soon as 5 d after ischemia already in the field CA 1 of the hippocampus itself. Astrocytes aggregated in the damaged area (fig. 5 c). There was no visible change in this aggregation from the 5th to the 20th d after ischemia. The rod shaped (micro-) glial cells obviously showed the same behaviour. But since they could not be clearly identified and some of them displayed the expression of GFAP, the proper affiliation of all or some rod like cells to the microglia group remains somewhat tentative. Yet, classical neuropathology because of the characteristic shape of these cells, would not doubt to consider them as microglia (fig. 5d). Discussion

The development of ischemic cell damage became clearer, when brains of animals exposed to hypoxic conditions could be well preserved by perfusion fixation: The different steps of ischemic cell change could be followed (BRIERLY 1984). It was found, that hypoxic change starts with cytoplasmic vacuolation and ends with homogenizing cell change. The latter only persists for days or weeks; damaged cells then disappear. Both in zones of selective vulnerability, e.g. in ammon's hom and in infarcts, ischemic cell changes were supposed to take place. Yet, KIRINO and SANO (1984) claimed from their findings in gerbils, that in some parts of ammon's hom, some kind of delayed necrosis of ganglion cell is met, if the damage is not too severe. Our findings in rats with territorial infarcts in the first model give - despite of some overlapping of the stages of development - evidence of roughly three main consecutive morphological alterations in areas of infarction: After one week, only pallor (Erbleichung) was seen: Perikarya had disappeared, but reactive cellular infiltration was weak or absent. After 2 weeks, there was a dense infiltration of cells in the center of the infarct, mostly by microglial Exp. Pathol. 35 (1988) 4

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cells. After 3 weeks, colliquation was going on or cysts had already been formed; the center of the infarct showed accumulated lipid phagocytes and the periphery a dense accumulation of astrocytes. This sequence is in agreement with our knowledge on the evolution of colliquation necrosis in the brain in territorial infarcts in man and experimental animals. It especially shows that in these infarcts, ischemic cell changes had taken place in the fIrst 7 d, since the zones of pallor were mostly devoid of perikarya. Only seldom some shrunken cells were yet found. In the second model - carotid occlusion and hypotension - the damage is, as previously shown by several authors, almost exclusively restricted to the CA 1 layer of hippocampus and to some hilar cells next to CA4. The same pattern of lesions was found in few instances in the fIrst model. In addition, in the first model, small territorial infarcts were seen, which included parts of the fIeld CA 1. Thus a direct comparison of CA 1 damage of different pathogenetic origin is possible. Three observations have been possible: First: Selective CA 1 changes follow the delayed course: There is no alteration during the first 2 d after ischemia; the ischemic shrinking starts after 3 or 4 d, but there was a wealth of obviously damaged, shrunken, homogenized cells after 1 and 2 weeks. A slight reduction of cell number only took place starting from the fIrst week after ischemia. Thus, delayed ischemic damage in CA 1 displays later onset and slower disappearance of ischemic cells. The same phenomenon concerning the long preservation of shrunken cells in ammon's hom could be observed in the fIrst model, if the ischemic sequelae were restricted to neurons of the hippocampus proper. Thus, this delayed ischemic cell change obviously occurs, if exclusively neurons are affected. In this case, the glial reaction also is different: Microglial cells do not change into fat granule cells and reactive astrocytes are eventually found in close connection to shrunken neuronal perikarya. Second: Small territorial infarcts develop through pallor zones devoid of neuronal perikarya to cysts containing fat granule cells and reactive astrocytes in the adjacent, partly preserved tissue. An intermediate stage is the accumulation of rod shaped microglial cells; the neuronal perikarya are with few exceptions - lacking after 1 week. Thus, classic ischemic cell change in territorial infarcts obviously follows a much faster course both concerning onset of shrinking and loss of neurons. In this case, microglial cells change into lipophages; astrocytes have no direct access to the ischemic neuronal somata. Third: This rapid change could be seen in the hippocampus, including CA 1, if this was part of small territorial infarcts. CA 1 neurons then were lost within one week, lipophages then appeared after accumulation of microglial cells and after three weeks already, a complete colliquated necrosis was formed. The type of cellular damage - slow or rapid - in our experiments was dependent upon the morphological appearance of the necrosis: if neuronal necroses were selective, they appeared as delayed, if they took place together with infarction of whole territories, they were rapid. A second obvious correlation from the morphological viewpoint is the close connection with astrocytes in the first and lipophages in the second instance. Several mechanisms have been thought to cause this delayed ischemic cell change in the field CA 1 of ammon's hom, which on the other hand was an outstanding example of classic selective

Fig. 3a. Ischemic cell changes in the ammon's hom. Most of the older cells are shrunken, hypochromatic and homogeneous. Few unaltered somata of nerve cells. In the right half of the picture, rod shaped cells (microglia) are seen as reaction to the cellular damage. Coelestin blue, X 500. Fig. 3 b. Accumulation of astrocytes in the layer CA 1 of ammon's hom. This band exactly corresponds to the damaged cells in fIg. 3 a. Cells, however, are not visible in the picture. GFAP, X 200. Fig. 3c. Accumulation of astrocytes densely packed in the field CA 3 of ammon's hom. On the right side one sees the end of the former ribbon of pyramidal cells. GFAP, X 125. Fig. 3d. Evidence of complete lack of ganglion cells in the affected area. On the right side on the bottom of the picture part of CA3, on the left side part of gyrus dentatus. Bodian, X 125. 226

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Fig. 3.

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Fig. 4a. Advanced cystic transformation of an infarct in the cerebellum. The cyst is almost completly formed. In the cyst fluid, some fat granule cells remain. In the top of the picture one sees the parallel glial fibers in the molecular part of the cerebellum. In this part the astroglial border reaction is very pronounced. GFAP, x 125. Fig. 4 b. Concentric arrangement of fat granule cells in the center and astrocytes in the periphery of the cyst. GFAP, x 125. 1987). Lately, there were indications that cells die by overexcitation al. 1987). This paper aims to reconsider the possible role ofthe amount of damage to only susceptible cells or whole territories and the different glial response to those events. In consequence, the role of various experimental arrangements for different types of human ischemia should be reconsidered. vulnerability

(NEDERGAARD

(J!2lRGENSEN et

Fig. 5 a. Complete destruction of ammon's horn with shrinking and hyperchromasia of ganglion cells and status spongiosus as well as cellular reaction. Cresylviolet, x 500. Fig. 5b. Forming of astrocytes in early stages of reaction to complete damage of ammon's horn. GFAP, X 500. Fig. 5c. Accumulation of cells near the partial reduction of pyramidal cell somata in ammon's horn. In the lower part of the picture one sees a well preserved ganglion cell of this layer. GFAP, X 500. Fig. 5 d. Most cells are damaged in ammon's horn. Pronounced reaction of rod like microglial cells next to vessels. Cresylviolet, x 200. 228

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Fig. 5.

16

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385-401. (Received May 20, 1988; Accepted May 30, 1988)

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