Temporal Relation of Calcium-Calmodulin Binding and Neuronal Damage After Global Ischemia in Rats

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1 876 Temporal Relation of Calcium-Calmodulin Binding and Neuronal Damage After Global Ischemia in Rats T.J. DeGraba, MD; P.T. Ostrow, MD; R.A. Strong, BS; R.M. Earls, BS; Z.J. Zong, MD; and J.C. Grotta, MD Background and Purpose: This study explores the temporal relation of the severity of ischemia and calcium-calmodulin binding in vulnerable and resistant brain regions in a commonly used model of global ischemia. Methods: Immunohistochemical assay of free calmodulin unbound to calcium and light microscopic histological damage were measured in rats after 5, 10, or 20 minutes of global ischemia. Results: After 24 hours of reperfusion, decreased calmodulin staining, representing increased calcium influx and calcium-calmodulin binding, correlated with increasing durations of ischemia across all brain regions. Based on a 4-point scale (4, extensive stain; 0, no staining), calmodulin staining after 5 minutes versus 10 minutes of ischemia was 3.2 versus 1.9, respectively (p<0.05) and after 10 minutes versus 20 minutes of ischemia was 1.9 versus 1.0, respectively (p<0.01). The CA1 region displayed the greatest sensitivity to ischemia. Similar but less dramatic results were seen after 2 hours of reperfusion. After 72 hours of reperfusion, histological damage closely correlated with calcium-calmodulin binding after variable durations of ischemia. A threshold of 10 minutes of ischemia was required to cause calciumcalmodulin binding and irreversible neuronal damage. Surviving neuronal populations showed recovery of calmodulin staining 7 days after ischemia, representing a return of free calmodulin and normal calcium homeostasis. Conclusions: These correlations between calcium-calmodulin binding, histological damage, and duration of ischemia support the causal role of calcium influx in global ischemic injury and suggest the need for very rapid intervention after ischemia if calcium-mediated damage is to be prevented. (Stroke 1992^3: ) KEY WORDS calcium calmodulin cerebral ischemia neuroprotectlon rats Ischemia-induced neuronal damage is characterized by an excessive increase in intracellular calcium (Ca 2+ ). x The binding of calcium to calmodulin (CaM), a Ca 2+ -binding protein, is a pivotal step in the regulation of neuronal function and activation of Ca/ CaM-dependent enzymes. Recent studies have implicated the involvement of these Ca/CaM-dependent phosphorylating enzymes in neuronal damage. 2 " 5 Calcium is a regulator of neuronal excitability and functions as a second messenger in many neuronal functions. In addition, synaptic vesicle modulation, cytoskeletal stability, and neurotransmitter synthesis and release are regulated by calcium-dependent protein phosphorylation Despite this information on cellular biochemical mechanisms, the precise sequence of events and From the Department of Neurology (TJ.D., J.C.G., RAS., R.M.E.), University of Texas Medical School at Houston, the Department of Pathology (P.T.O.), State University of New York at Buffalo, and Beijing Neurosurgical Institute (ZJ.Z.) at Beijing, China. Supported in part by National Institutes of Health grant RO-1 NS to J.C.G. Address for correspondence: Thomas J. DeGraba, MD, Department of Neurology, University of Texas School of Medicine, 6431 Fannin, Suite MSB, Houston, TX Received July 26, 1991; accepted February 9, mechanism of neuronal death remain elusive. Further understanding of the response of the CaM-activated enzyme system to varying durations of ischemia may help to determine the events leading to neuronal damage. It is also important to understand the pattern of recovery of Ca 2+ homeostasis and its dependent enzyme activity in surviving neurons. Because of the perceived importance of calcium in the genesis of neuronal injury, clinical studies are now being designed to intervene with calcium and glutamate antagonists to prevent calcium influx into neurons after stroke. 11 " 21 An understanding of the temporal relation between Ca 2+ influx, Ca-CaM binding, and neuronal damage is critical to the design of these studies because it will determine the time window within which therapy must be started and the duration of therapy. The present study explores the correlation of Ca- CaM binding to duration of ischemia and neuronal damage and is preliminary to analysis of the CaMactivated enzymes calcium/calmodulin-dependent protein kinase II (CaM kinase II) and protein kinase C after ischemia. Picone et al 21 developed an immunohistochemical assay using an antibody specific for free CaM. It was demonstrated that severe global ischemia (20 minutes) in rats results in Ca 2+ influx and Ca-CaM binding in hippocampal neurons during the first 24

2 DeGraba et al > & Calcium-Calmodulin Binding 877 FIGURE 1. Photomicrograph in which calmodulin (CaM) staining of normal rat hippocampus is represented by dark coloration of individual neurons. The CaM-antibody binding to free CaM in all cells, including CAl (thick arrow) and dentate (thin arrow), is characteristic of the nonischemic state. x55. hours of reperfusion before recognizable histological damage. We took advantage of this unique antibody to further study the characteristics of Ca-CaM binding and Ca2+ influx in various vulnerable and resistant brain regions after briefer durations of global ischemia. We also extended our period of measurement to 7 days to evaluate recovery in surviving neuronal populations. Materials and Methods Male Wistar rats ( g) were subjected to global ischemia using an adaptation of the four-vessel occlusion model. Coagulation of the vertebral arteries was performed under chloral hydrate anesthesia, and loose ligatures were placed on the carotid arteries for future rapid access. Twenty-four hours later, after a few seconds of ether inhalation, nontraumatic clips were placed on the common carotid arteries, and the cervical muscles were ligated for 5, 10, or 20 minutes. Heating lamps and warming blankets were used to maintain rectal and scalp temperature at 37 C. Electroencephalographic (EEG) activity was monitored to ensure duration and effectiveness of the global ischemia. Rats that did not display a complete loss of EEG rhythm or had a postischemic seizure were not included in the analysis. Preparation of brain slices was carried out for CaM immunohistochemical staining as previously described.22 Brain sections of groups of six rats exposed to 5,10, or 20 minutes of ischemia after 2 hours, 24 hours, or 7 days of reperfusion were incubated in sheepinduced CaM antibody, which binds specifically to free CaM (i.e., the CaM not bound to Ca2+ and target protein). The sections were incubated with rabbit antisheep peroxidase-conjugated secondary antibody, which binds to and stains only CaM-antibody complex. Thus, only normal cells with free CaM will stain, and ischemic cells with Ca2+-activated CaM-target protein complex do not stain. Sets of postischemic and normal brain were processed simultaneously to ensure comparable staining. Intensity of staining in the dorsal CAl, lateral CA3, endal limb of dentate, and layers I-VI of the parietal cortex was graded on a 4-point scale (4, extensive staining of normal neuronal soma; 3, some staining of soma; 2, some staining but soma not distinguishable; 1, minimal staining; 0, no staining) (Figures 1 and 2). The evaluator was blinded to the duration of ischemia and the period of reperfusion. Statistical analysis was performed using Kruskal-Wallis one-way analysis of variance (ANOVA) by rank. The specificity of the CaM antibody to free CaM was demonstrated by in vitro immunohistochemical dot staining of CaM incubated in ethylene glycol tetraacetic acid (EGTA), Ca2+, and Ca2+ with brain homogenate. Staining only occurred in the absence of Ca2+ and target protein.21 Quantification of CaM by Western blot technique revealed no change between control and ischemic rats.21 Calmodulin overlay gel autoradiography also demon-

3 878 Stroke Vol 23, No 6 June 1992 FIGURE 2. Photomicrograph in which a significant decrease in staining is seen in vulnerable CAl region (thick arrow) compared with dentate (thin arrow) after 10 minutes of global ischemia and 24 hours of reperfusion. Loss of staining is also marked at 10 minutes of ischemia in CAl region compared with nonischemic animals (see Figure 1). Loss of staining represents calcium influx with calcium-calmodulin binding to target protein, thus preventing antibody binding of calmoduun and staining normally seen in nonischemic neurons. Note that staining of dentate is relatively unchanged despite ischemic injury to CAl region. x55. strated no degradation of target protein immediately after ischemia or after 2 and 24 hours of reperfusion. To confirm CaM binding to target protein after ischemia, availability of CaM binding sites was measured by incubation of hippocampal and CAl slices of rats exposed to 20 minutes of ischemia in CaM labeled with Bolton-Hunter iodine-125 (CaM 1-125) with either 1 mm CaCl or 1 mm EGTA. Sections were incubated overnight, washed in buffer, and the CaM binding determined by gamma activity. CaM-I-125 binding in ischemic rats was one tenth that of control rats, suggesting that CaM remains bound to target protein postischemia. Seventy-two hours after 5, 10, or 20 minutes of ischemia, while under ether anesthesia, six rats from each group and six control rats were perfused with phosphate-buffered 10% formalin, decapitated, and the brains embedded in paraffin to be sectioned and stained with hematoxylin and eosin. Ischemic histological neuronal changes were quantitated in CAl, CA3, dentate, and parietal cortex by light microscopy. Regions were scored from 0 to 4 based on the percentage of cells with shrunken eosinophilic cytoplasm and pyknotic nuclei (0, 0%; 1, 1-25%; 2, 26-50%; 3, 51-75%; 4, %). Evaluation was again blinded as to the duration of ischemia, and group differences were evaluated by Kruskal-Wallis one-way ANOVA. Results Loss of CaM staining, representing Ca2+ influx and Ca-CaM binding, correlated with increasing durations of ischemia across all brain regions (Figure 3, Table 1). CalmoduUn staining 24 hours after 5 minutes versus 10 minutes of ischemia was 3.2 versus 1.9, respectively (p<0.05), and after 10 minutes versus 20 minutes of ischemia was 1.9 versus 1.0, respectively (p<0.01). The CAl region and the cortex displayed the greatest reduction of CaM staining in response to the increasing durations of ischemia. Similar but much less severe reduction was found after 2 hours of reperfusion (Table 1). Immunohistochemical staining of CaM was also performed at 7 days (Table 1). The CAl region showed persistent loss of CaM staining after 20 minutes of ischemia, whereas shorter ischemic intervals (5 and 10 minutes) resulted in a return toward normal Ca2+ homeostasis by 7 days (Figure 4, top). In contrast, the cell populations that demonstrated a less significant early response of Ca2+ influx to ischemia, such as the dentate, showed recovery of CaM staining by 7 days even after 20 minutes of ischemia (Figure 4, bottom and Table 1). The histological score demonstrated worse damage after 20 minutes versus 10 minutes of ischemia across all brain regions at 72 hours of reperfusion (2.4 versus 1.4,

4 DeGraba et al Calcium-Calmodulin Binding 879 Global Ischemia 24 Hrs Reperfusion FIGURE 3. Bar graph showing calmodulin (CAM) staining after 24 hours of reperfusion in all regions combined, CA1, CA3, dentate, and cortex, demonstrating a graded response to increasing durations of ischemia (5, 10, and 20 minutes). CA1 and cortex showed the greatest vulnerability to ischemia with a significant decrease in staining between 5 and 10 minutes of ischemia (p<0.025). Grading score of 4, normal staining; 0, no staining ' 201 S All Regions * <.O5 * * c.025 W SB1 CA-1 t t S 10" 201 CA-3 S 10' 20' s c.01 <.0O5 respectively; /?<0.005) (Table 2). Again, the most significant neuronal damage was seen in the CA1 region, with >85% ischemic neurons after 20 minutes of ischemia (Table 2, Figure 5). In comparison with other neuronal populations, the CA1 showed significantly more damage after shorter durations of ischemia. After 10 minutes of ischemia, the CA3, dentate, and cortex displayed <25% ischemic neurons, whereas the CA1 had >65% (Table 2). Histological damage correlated very closely with CaM staining, best seen in the CA1 region (Figure 5). An increase in the percentage of ischemic neurons seen after 72 hours of reperfusion corresponds to a decrease in CaM staining. An apparent threshold of Ca2+ influx to ischemic duration is represented by the change in CaM staining in all regions seen at 5 versus 10 minutes of ischemia (3.2 versus 1.9, p<0.05) (Figure 3). This threshold phenomenon can be seen by examining CA1 CaM staining in individual rats (Figure 6). In all rats, neurons revealed near normal staining after 5 minutes of ischemia and severe loss of CaM staining after 20 minutes of ischemia. A variable response, however, was seen after 10 minutes of ischemia; some rats displayed near nortable 1. 10' 2tr Cortex Dentate mal staining while others showed severe loss of staining, suggesting a threshold of Ca-CaM binding to target protein in vulnerable neurons at 10 minutes of ischemia. This threshold can also be appreciated by examining the correlation between CaM staining and histological damage in all regions observed as well as the CA1 mentioned above. Substantial Ca-CaM binding (CaM staining <1) corresponds to >75% ischemic neurons seen at 72 hours of reperfusion as well as persistent loss of CaM staining at 7 days. Discussion Through the use of a unique antibody to free CaM and consequently with the ability to measure Ca-CaM binding, our data support the causal role of Ca2+ influx and Ca/CaM-mediated events in the production of neuronal damage. Furthermore, the degree of Ca-CaM binding is a predictor of the severity of neuronal damage after ischemia. In vitro immunohistochemical blot studies showed that in the presence of calcium, CaM rapidly binds to target protein, making it unavailable for staining.22 In Calmodulin Staining Score of Groups of Rats (n=6) After 5, 10, or 20 Minutes of Ischemia Reperfusion 5 minutes of ischemia 2 hours 24 hours 7 days 10 minutes of ischemia 2 hours 24 hours 7 days 20 minutes of ischemia 2 hours 24 hours 7 days All regions CA1 CA3 Dentate Cortex 3.29± ± ± ± ± O± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.30 Values are mean±sem. Score based on 4-point scale of 4, extensive staining of normal neuronal soma; 3, some staining of soma; 2, some staining but soma not distinguishable; 1, minimal staining' 0, no staining.

5 880 Stroke Vol 23, No 6 June 1992 Calmodulin Staining: CA-1 CO 'c "3 55 o "5 CD D ( «\ * # 5 minutes A 10 minutes 20 minutes 2 hr 24 hr 7d Reperfusion Calmodulin Staining: Dentate FIGURE 4. Line graphs showing calmodulin staining measured after 2 hours, 24 hours, and 7 days of reperfusion. Top panel: Partial recovery of calmodulin staining after 5 and 10 minutes of ischemia in CA1 region represents a return of normal Ca 2+ homeostasis in surviving neurons by 7 days. Severe ischemia (20 minutes) results in extensive calcium-calmodulin binding and no return of staining after 7 days. This is believed to be a result of irreversible neuronal damage by this period of time. Bottom panel: Substantial recovery of calmodulin staining was seen after 20 minutes of ischemia in dentate region, representing a population of neurons more resistant to severe global ischemia. Note that staining score does not fall below a score of 1 at 24 hours, as it does in CA1 region. Grading score of 4, normal staining; 0, no staining. CAM, calmodulin. 5 minutes A 10 minutes 20 minutes 2hr 24 hr 7d Reperfusion the absence of Ca 2+ (using EGTA), CaM is freely available for immunohistochemical staining. Therefore, loss of neuronal CaM staining represents increased intracellular Ca 2+ and Ca-CaM binding. Although free Ca 2+ may become available to CaM from sequestered intracellular stores, a major source of free neuronal Ca 2+ is believed to be extracellular. This conclusion is based on studies demonstrating preservation of CaM staining with pharmacological agents that block the influx of extracellular Ca 2 *. 22 " 24 Our studies demonstrate that with increasing durations of ischemia, there is a corresponding increase of Ca 2+ influx and Ca-CaM binding. The concept that the degree of Ca 2+ influx is directly linked to the duration of ischemia is important in postulating Ca 2+ as a mediator of neuronal injury. It highlights the fact that events leading to neuronal death begin early in the ischemic insult and are time dependent. After ^10 minutes of ischemia, significant Ca-CaM binding can be found in vulnerable brain regions by 2 hours and is maximal by 24 TABLE 2. Histological Score of Groups of Rats (n=6) After 5, 10, or 20 Minutes of Ischemia and 72 Hoars of Reperfusion Duration of ischemia (min) All regions 1.05± ± ±0.32 CA1 1.86± ± ±0.11 CA3 0.42± ± ±0.55 Dentate 0.50± ± ±0.47 Cortex 1.00± ± ±0.25 Values are mean±sem. Score determined by hematoxylin and eosin staining, based on percentage of cells with shrunken eosinophilic cytoplasm and pyknotic nuclei (0, 0%; 1, 1-25%; 2, 26-50%; 3, 51-75%; 4, %).

6 DeGraba et al Calcium-Calmodulin Binding 881 Histological Damage vs Calmodulin Staining CA-1 FIGURE 5. Line graph showing histological damage (A A), represented by percentage of ischemic neurons seen at 72 hours of reperfusion, compared with calmodulin staining ( ) at 24 hours in CA1 region. Increase in percentage of ischemic neurons correlates closely with increasing Ca2* influx, represented by loss of calmodulin staining seen after 5, 10, and 20 minutes of global ischemia. Grading score (calmodulin staining) of 4, normal staining; 0, no staining. See text for explanation of histological score. CAM, calmodulin. 4.0 Minutes of Ischemia hours. This underscores the need to intervene with neuroprotective agents at the earliest possible time after an ischemic episode. A threshold phenomenon appears to exist with respect to the duration of ischemia and Ca2+ influx. Although subtle, the greatest loss of CaM staining in vulnerable brain regions (CA1, CA3, and cortex, but not dentate) occurs after ischemic intervals of 5-10 minutes in duration. This finding suggests numerous possibilities. It may represent the time required for excitatory amino acids to maximally activate JV-methyl-D-aspartate receptor-operated Ca2+ channels. It may also represent the time required to reach a concentration of intracellular free Ca2+ at which CaM binding sites become saturated. Finally, it may represent the time required for intracellular free Ca to exceed the cell's homeoglobal Ischemia 24 Hrs Reperfusion CA-1 p <.025 p-ns «- - - CO o Si 8' 10* 2ff Minutes of Ischemia FIGURE 6. Graph showing calmodulin staining of CA1 region after 24 hours of reperfusion, represented as individual score ( ) and mean (boxes). Near normal staining is seen in all rats exposed to 5 minutes of ischemia, and severe loss of staining is seen in all rats after 20 minutes of ischemia. A variable response is seen after 10 minutes, suggesting a threshold ofca2+ influx at that duration of ischemia. Grading score of 4, normal staining; 0, no staining. CAM, calmodulin. static mechanisms. These possibilities remain to be explored. It should be emphasized that the time relations between Ca-CaM binding and the duration of ischemia and reperfusion described in this study apply to global ischemia and may not be true after focal ischemia. Similar studies in a middle cerebral artery occlusion model of focal ischemia are presently under way in our laboratory. Equal in importance to the correlation of Ca-CaM binding and duration of ischemia is the close regional correlation between Ca-CaM binding and subsequent neuronal damage. Beyond a critical level of Ca-CaM binding at 24 hours, neurons exhibit irreversible cell damage, seen by hematoxylin and eosin stain at 72 hours and the lack of CaM staining at 7 days. This is best seen in the CA1 region after 20 minutes of ischemia. The amount of Ca-CaM binding at 24 hours predicted the likelihood of >50% neuronal damage after 10 and 20 minutes of ischemia (Figure 5). These data refute the notion that Ca2+ influx is a nonspecific epiphenomenon of ischemia. The correlation of Ca-CaM binding and histological damage suggests at least one major avenue for investigating the mechanism of irreversible neuronal damage. Calcium-activated CaM interacts with a number of enzymes, most notably CaM kinase II, which has important regulatory functions including the activation of genes involved in protein synthesis.25 Previous results from our laboratory have shown that Ca-CaM binding after ischemia correlates closely with loss of CaM kinase II activity believed to be due to translocation of the enzyme into the particulate fraction of the cell.26 Return of CaM staining in surviving neurons parallels return of CaM kinase II activity by 7 days after ischemia.26 Finally, hippocampal cells show selective vulnerability to ischemia and have high concentrations of CaM kinase II compared with other brain regions.27'28 Further studies of this association are under way in our laboratory. In this study, after 20 minutes of ischemia there was no recovery of CaM staining by 7 days in vulnerable CA1 neurons. Previous observations reveal extensive

7 882 Stroke Vol 23, No 6 June 1992 gliosis by hematoxylin and eosin throughout the CA1 region with few if any surviving neurons present, weeks after 20 minutes of global ischemia. 23 Therefore, the lack of recovery of CaM staining at 7 days is undoubtedly due to complete destruction of neurons by this time and does not represent cell survival with a persistent loss of free CaM availability. At 7 days of reperfusion, after 5 and 10 minutes of ischemia the CaM staining score of 3 represents normal cell morphology and staining with only a mild decrease in the number of surviving neurons. As seen in the CA1 region after 5 and 10 minutes of ischemia, cells destined to recover will reestablish normal Ca 2+ homeostasis by 7 days. Dentate neurons show the most resistance to loss of CaM staining, the fastest recovery of normal CaM staining, and the least histological damage. Further evaluation of the differences between vulnerable and resistant neuron populations may provide insight into the mechanism of neuronal damage. Finally, recovery of normal calcium homeostasis between 24 hours and 7 days suggests that "neuronal-protective" therapies should be administered for up to but not necessarily exceeding 7 days. In conclusion, our results support a causal role of Ca 2+ influx and Ca/CaM-mediated events in the production of neuronal damage. Recognition of early changes in Ca-CaM binding supports the need for rapid intervention after acute ischemic insults. Finally, the pattern of Ca 2+ influx and homeostasis, along with its functional interaction with CaM, adds another piece to the puzzle of the mechanism of ischemia-induced neuronal death. Acknowledgments Thanks to Howard Rhoades, MD, Department of Psychiatry, University of Texas Medical School at Houston, for statistical assistance, and to Sozos C. 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