- Email: [email protected]
Digoxin antibody prevents cerebral hemorrhage-induced hypertension
AJH
2003; 16:1062–1065
Digoxin Antibody Prevents Cerebral Hemorrhage-Induced Hypertension Joa˜ o C. Menezes and Varujan Dichtchekenian Background: Brain injury may induce hypertension. Because serum ouabain-like compound (OLC) has vasoconstrictor activity, digoxin antibody antihypertensive effects were evaluated using an intracerebroventricular (ICV) hemorrhage rat model. Methods: Four ICV infused Wistar rat groups were studied: control; blood; blood plus digoxin antibody, and cerebrospinal fluid-like solution. Tail-cuff blood pressure, cumulative sodium balance, and serum OLC were measured. Results: The ICV blood infusion increased blood pressure (BP) and OLC without sodium balance change.
H
Digoxin antibody prevented BP and OLC rise. Blood pressure was positively correlated with OLC in blood and blood plus digoxin antibody rats (R ⫽ 0.63; P ⬍ .05). Conclusions: Cerebral hemorrhage increased OLC and BP, which were reversed by digoxin antibody administration. Am J Hypertens 2003;16:1062–1065 © 2003 American Journal of Hypertension, Ltd. Key Words: Cerebral hemorrhage, digoxin antibody, hypertension, ouabain-like compound, rat.
igh blood pressure (BP) is one of the most potent risk factors for the first and recurrent stroke.1 Intracerebral hemorrhage represents approximately 10% of all strokes with higher risk of fatality.2 Blood pressure control for primary prevention, using the current antihypertensive therapies,3 and for secondary prevention in ischemic stroke, using perindopril,4 is well established. In acute stroke, BP elevation may occur in 80% of the patients on the day of admission to the hospital and then it decreases to 30% in the next few days.5 The management of BP in acute hemorrhagic stroke is controversial because aggressive BP reduction could lead to clinical worsening, due to cerebral perfusion failure. Sympathetic6 and reninangiotensin system7 overactivity are some of the mechanisms proposed. Another substance, an ouabain-like compound (OLC), probably originating in the central nervous system, was initially described as the reason for intravascular volume expansion.8 Acting as an Na-K-ATPase inhibitor, it could have vasoconstrictor activity by increasing the intracellular calcium.9 In patients with subarachnoid hemorrhage,10 as well as in animal models with intracerebroventricular (ICV) manipulations,6 an overproduction of this substance by local stimulation was observed. Yamada et al11 blocked the OLC effect on
natriuresis using ICV digoxin antibody. Data from our previous studies showed high levels of OLC in salt-sensitive hypertensive humans.12 Based on the fact that an OLC with inhibitory activity on vascular Na-K-ATPase and therefore, vasoconstrictor activity, could be overproduced after brain injury, we hypothesized that the blockade of this substance with digoxin antibody would be beneficial in this circumstance.13 This study was conducted to determine whether an OLC is involved in BP changes of awake animals submitted to ICV hemorrhage. In addition, the effects of ICV infusion of digoxin antibody as a therapeutic approach were evaluated.
Received July 22, 2003. First decision August 5, 2003. Accepted August 27, 2003. From the Basic Research Laboratory, Nephrology (LIM-12), Medicine School, Sao Paulo University, Sao Paulo, Brazil. This work was supported by Fundac¸a˜o de Amparo a Pesquisa do
Estado de Sa˜o Paulo, FAPESP. Address correspondence and reprint requests to Dr. Varujan Dichtchekenian, Faculdade de Medicina da Universidade de Sao Paulo Av. Dr. Arnaldo, 455 3, andar-sala 3310 Cerqueira Ce´sar, Sao Paulo, Brasil CEP 01246 903; e-mail: [email protected]
0895-7061/03/$30.00 doi:10.1016/j.amjhyper.2003.08.001
Methods Animals Male Wistar rats weighing 250 to 350 g were used. Ad libitum diet containing 0.6 Na g/100 mL and tap water was given. The experimental protocol was approved by the animal studies ethical committee of Sao Paulo University Medical School. Surgical Implant of the Cannula A skin incision, under anesthesia with thiopental sodium (50 mg/kg intraperitoneally) was performed, and a stain-
© 2003 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
AJH–December 2003–VOL. 16, NO. 12
DIGIBIND DECREASES BP IN CEREBRAL HEMORRHAGE
1063
Table 1. Tail– cuff BP, BP increase, cumulative sodium balance, and serum inhibitory activity on Na-KATPase Blood Pressure (mm Hg) D2 Blood Blood/ab CSF-like Control
117 125 128 100
⫾ ⫾ ⫾ ⫾
D4 6 3 6 1b
125 123 127 101
⫾ ⫾ ⫾ ⫾
D6 3 6 4 2
125 120 113 102
⫾ ⫾ ⫾ ⫾
D11 5 4 2 1
132 119 110 103
⫾ ⫾ ⫾ ⫾
a
2 4 2 3
⌬BP (mm Hg)
CSB (mEq Na/100g/6d)
IA% (%)
D11–D2
D6–D11
D11
14 ⫺7 ⫺19 3
⫾ ⫾ ⫾ ⫾
c
6 5 7 2
1.8 1.2 1.5 2.0
⫾ ⫾ ⫾ ⫾
0.1 0.2 0.4 0.5
19 ⫺13 ⫺72 ⫺19
⫾ ⫾ ⫾ ⫾
15d 25 7 3
a
P ⬍ .05 D2 v D11. b P ⬍ .05 control v blood, blood/ab, and CSF-like. c P ⬍ .05 blood v blood/ab, and CSF-like. d P ⬍ .05 blood v CSF-like. D ⫽ day; D2, D4 ⫽ before ICV infusions; D6, D11 ⫽ after ICV infusions; ⌬BP ⫽ D11–D2; CSB ⫽ cumulative sodium balance from D6 to D11; IA% ⫽ inhibitory activity of serum on Na-K-ATPase. Groups: Blood ⫽ ICV hemorrhage (n ⫽ 6); blood/ab ⫽ ICV hemorrhage/ ICV digoxin antibody (n ⫽ 5); CSF-like ⫽ ICV cerebrospinal-like solution (n ⫽ 6); Control ⫽ no ICV cannula or infusions (n ⫽ 5).
less steel cannula was introduced stereotaxically down to the right lateral ventricle. Brain histology was performed to confirm the site of ICV infusion. Animal Groups and ICV Infusions Group 1 animals were ICV infused with autologous venous blood, through an infusion pump at the rate of 50 L/12 min (n ⫽ 6). Group 2 animals were ICV infused with Fab fragments of digoxin antibody (DIGIBIND, Glaxo-Smith-Kline, Victoria, Australia; 10 mg/mL) just before ICV blood infusion at the rate of 10 L/2.4 min (n ⫽ 5). Group 3 animals were ICV infused with a cerebrospinal fluid (CSF)-like solution containing: Na⫹ 140 mEq/L, K⫹ 3 mEq/L, Ca2⫹ 1.3 mEq/L, Mg2⫹ 1 mEq/L, HCO3 25 mEq/L, and glucose 60 mg/dL, at a rate of 50 L/12 min (n ⫽ 6). Group 4 (control group) was anesthetized and sham operated on the same day of cannula introduction and ICV infusions (n ⫽ 5). Laboratory Measurements One week after cannula implant, the animals were placed in metabolic cages for an adaptation period of 3 days, and subsequently maintained for 11 days. The ICV infusions were performed on the fourth day under anesthesia (thiopental sodium 50 mg/kg intraperitoneally). Collections performed on the fifth day were discarded due to the influence of the anesthesia. Tail-cuff BP was measured on days 2, 4 (before), 6, and 11 (after) ICV infusions, six times on each day. Daily chow ingestion and urinary sodium excretion were measured to determine cumulative sodium balance. Urinary sodium concentration was measured with a flame photometer (CELM FC-280, Sao Paulo, Brazil). On the last day (day 11) before killing, blood samples were collected by cardiac puncture, under anesthesia, to measure the effects on Na-K-ATPase activity in renal outer medulla homogenate of normal rats. The [32 P]ATP hydrolysis method was used to determine enzyme activity.14
Na-K-ATPase Assay and Serum Inhibitory Activity on Normal Rat Renal Na-K-ATPase Activity Under anesthesia, normal rats were nephrectomized. Kidneys were sliced longitudinally, and outer medulla separated and homogenized for Na-K-ATPase assay. Four solutions were prepared to avoid influence of any other enzyme activity (solution A: 20 mmol/L KCl; solution B: 20 mmol/L KCl ⫹ 100 L of ouabain [0.018 g/mL; Sigma, St. Louis, MO]; solution C: no KCl ⫹ 100 L of ouabain; and solution D: 20 mmol/L KCl ⫹ 100 L of filtered animal serum). To estimate the effects of animal serum on Na-KATPase activity, comparison of maximal enzymatic activity (solution A) with the difference between solutions D and C were performed. Inhibition or stimulation of the enzyme was calculated. Statistical Analysis The analysis of variance test was used to compare independent groups. Blood pressure was analyzed with repeated measures (ANOVA). Spearman test was used for correlation. P ⬍ .05 was statistically significant. Results were expressed as mean ⫾ SEM.
Results Tail-cuff BP Table 1 shows tail-cuff BP. Between days 2 and 11 the following changes were observed: ICV blood infusion increased BP (P ⬍ .05), which could be prevented (P ⬎ .05) by digoxin antibody; CSF-like solution infusion decreased BP (P ⬍ .05) and control rats did not change BP (P ⬎ .05). Control animals presented lower BP before infusion (day 2, P ⬍ .05) compared to the other three groups. The BP difference (⌬BP) between day 11 and day 2 showed higher increase in ICV blood infused compared with ICV blood plus antibody and CSF-like rats (P ⬍ .05).
1064
DIGIBIND DECREASES BP IN CEREBRAL HEMORRHAGE
Cumulative Sodium Balance Table 1 shows cumulative sodium balance after ICV infusions in the four groups. No statistical difference was observed between groups (P ⬎ .05). Serum Inhibitory Activity on Na-K-ATPase Activity The percent inhibition (positive value) or stimulation (negative value) of rat serum on Na-K-ATPase activity of the outer medulla of normal rats is shown in Table 1. Serum of ICV blood rats showed Na-K-ATPase activity inhibition that was reversed by digoxin antibody to stimulation in ICV blood plus antibody rats. Serum of CSF-like and control animals showed stimulation of Na-K-ATPase activity. A significant difference between ICV blood and CSF-like animals was observed (P ⬍ .05). Correlation Between BP increase (⌬BP mm Hg) and serum inhibitory activity on Na-K-ATPase activity (IA%) A positive correlation between ⌬BP and IA% in ICV blood and ICV blood plus antibody groups, with R ⫽ 0.63 (P ⬍ .05) was observed. The ICV blood rats presented higher BP and IA% increase compared with ICV blood plus antibody rats.
AJH–December 2003–VOL. 16, NO. 12
in intracranial pressures to explain the BP increase. The type of ICV infused fluid and its effects on BP were analyzed and showed that blood, but not CSF-like solution, induced hypertension. Finally, according to Blaustein et al,9 endogenous OLC could inhibit vascular smooth muscle cell sodium pump increasing cytosolic sodium concentration and exchanging it for calcium. The high cytosolic calcium would increase vascular peripheral reactivity and BP. Digoxin antibody, used for digitalis intoxication treatment,13 was used based on its cross-reactivity with the endogenous hormone. The mechanism by which ICV infusion of digoxin antibody reduced BP is unknown. Hypothalamic or even vascular (the blood– brain barrier could be ruptured) action on the endogenous ouabain may explain these beneficial effects. In conclusion, cerebral hemorrhage increased BP through sodium pump inhibition. Digoxin antibody prevented BP increase.
Acknowledgments We thank the following people and institutions: 1) Dr. Antonio C Seguro and Dr. Lui Yu, Sao Paulo University, School of Medicine; 2) Hospital Israelita Albert Einstein; and 3) FAPESP, Fundac¸ a˜ o de Amparo a Pesquisa do Estado Sa˜ o Paulo.
Discussion An endogenous OLC with inhibitory activity on vascular Na-K-ATPase may be overproduced after brain injury, inducing an increase in BP. Therefore, we hypothesized that blockade of this substance with digoxin antibody may reduce BP after brain injury. Results confirmed this hypothesis. The ICV blood infusion increased BP and serum inhibitory Na-K-ATPase activity, which could be reversed with ICV digoxin antibody. The positive correlation between BP increase and Na-K-ATPase activity in ICV blood and ICV blood plus antibody confirmed these results. Available data in the literature indicate that cerebral manipulations induce, acutely, high intracranial pressure and BP, as seen in increased CSF sodium concentration and subarachnoid hemorrhage models.11,15 In this study, before ICV infusions (day 2), all three cannulated groups presented higher (P ⬍ .05) BP compared to the control rats. It is possible that the stress induced by cannulation could explain this higher baseline BP. On the other hand, ICV blood rats did not have an increase in BP (during the first 48 h). Instead, they have elevated BP 7 days after ICV infusion. Thus, it is unlikely that this late BP increase occurred due to experimental manipulations because control rats did not have an increase in BP and, on the contrary, CSF-like animals had decreased BP. Brain histology, performed 7 days after ICV infusions, did not show ventricle size increase, suggesting no later changes
References 1.
2. 3.
4.
5. 6.
7.
8.
9.
Donnan GA, Davis SM, Thrift A: The role of blood pressure lowering before and after stroke. Curr Opin Neurol 2003;16:81– 86. Labovitz DL, Sacco RL: Intracerebral hemorrhage: update. Curr Opin Neurol 2001;14:103–108. Perry HM Jr, Davis BR, Price TR, Applegate WB, Fields WS, Guralnik JM, Kuller l, Pressel S, Stamler J, Probstfield JL: Effect of treating isolated systolic hypertension on the risk of developing various types and subtypes of stroke: the Systolic Hypertension in the Elderly Program (SHEP). JAMA 2000;284:465–471. PROGRESS Collaborative Group: Randomised trial of a perindopril-based blood-pressure-lowering regimen among 6,105 individuals with previous stroke or transient ischaemic attack. Lancet 2001; 358:1033–1041. Loyke HF: The three phases of blood pressure in stroke. South Med J 1990;83:660 –663. Huang BS, Leenen FHH: Brain ouabain-like activity and the sympathoexitatory and pressor effects of central sodium in rats. Circ Res 1992;71:1059 –1066. Huang BS, Leenen FHH: Brain renin-angiotensin system and ouabain-induced sympathetic hyperactivity and hypertension in Wistar rats. Hypertension 1999;34:107–112. de Wardener HE, Mills IH, Clapham WF, Hayter CJ: Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin Sci 1961;21:249 – 258. Blaustein MP, Juhaszova M, Golovina VA: The cellular mechanism of action of cardiotonic steroids: a new hypothesis. Clin Exp Hypertens 1998;20:691–703.
AJH–December 2003–VOL. 16, NO. 12
10. Wijdicks EFM, Vermeulen M, van Brummelen P: Digoxin-like immunoreactive substance in patients with aneurysmal subarachnoid hemorrhage. Br Med J 1987;294:729 –732. 11. Yamada K, Goto A, Nagoshi H, Hui C, Omata M: Role of brain ouabain like compound in central nervous system-mediated natriuresis in rats. Hypertension 1994;23:1027–1031. 12. Dichtchekenian V, Gisiger S, Quental I, Santos SR, Marcondes M, Heimann JC: Higher salt consumption, digoxin-like factor, and nifedipine response are associated with salt sensitivity in essential hypertension. Am J Hypertens 1992;5:707–712.
DIGIBIND DECREASES BP IN CEREBRAL HEMORRHAGE
1065
13. Boucher BA, Lalonde RL: Digoxin-specific antibody fragments for the treatment of digoxin intoxication. Clin Pharm 1986;5: 826 –827. 14. Woolfson RG, Poston L, de Wardener HE: Dioxin-like inhibitors of active sodium transport and blood pressure: the current status. Kidney Int 1994;46:297–309. 15. Verlooy J, van Reempts J, Haseldonckx M, Borgers M, Selosse P: Hemodynamic, intracranial pressure and electrocardiographic changes following subarachnoid hemorrhage in rats. Acta Neurochir 1992;115: 118 –122.
2003; 16:1062–1065
Digoxin Antibody Prevents Cerebral Hemorrhage-Induced Hypertension Joa˜ o C. Menezes and Varujan Dichtchekenian Background: Brain injury may induce hypertension. Because serum ouabain-like compound (OLC) has vasoconstrictor activity, digoxin antibody antihypertensive effects were evaluated using an intracerebroventricular (ICV) hemorrhage rat model. Methods: Four ICV infused Wistar rat groups were studied: control; blood; blood plus digoxin antibody, and cerebrospinal fluid-like solution. Tail-cuff blood pressure, cumulative sodium balance, and serum OLC were measured. Results: The ICV blood infusion increased blood pressure (BP) and OLC without sodium balance change.
H
Digoxin antibody prevented BP and OLC rise. Blood pressure was positively correlated with OLC in blood and blood plus digoxin antibody rats (R ⫽ 0.63; P ⬍ .05). Conclusions: Cerebral hemorrhage increased OLC and BP, which were reversed by digoxin antibody administration. Am J Hypertens 2003;16:1062–1065 © 2003 American Journal of Hypertension, Ltd. Key Words: Cerebral hemorrhage, digoxin antibody, hypertension, ouabain-like compound, rat.
igh blood pressure (BP) is one of the most potent risk factors for the first and recurrent stroke.1 Intracerebral hemorrhage represents approximately 10% of all strokes with higher risk of fatality.2 Blood pressure control for primary prevention, using the current antihypertensive therapies,3 and for secondary prevention in ischemic stroke, using perindopril,4 is well established. In acute stroke, BP elevation may occur in 80% of the patients on the day of admission to the hospital and then it decreases to 30% in the next few days.5 The management of BP in acute hemorrhagic stroke is controversial because aggressive BP reduction could lead to clinical worsening, due to cerebral perfusion failure. Sympathetic6 and reninangiotensin system7 overactivity are some of the mechanisms proposed. Another substance, an ouabain-like compound (OLC), probably originating in the central nervous system, was initially described as the reason for intravascular volume expansion.8 Acting as an Na-K-ATPase inhibitor, it could have vasoconstrictor activity by increasing the intracellular calcium.9 In patients with subarachnoid hemorrhage,10 as well as in animal models with intracerebroventricular (ICV) manipulations,6 an overproduction of this substance by local stimulation was observed. Yamada et al11 blocked the OLC effect on
natriuresis using ICV digoxin antibody. Data from our previous studies showed high levels of OLC in salt-sensitive hypertensive humans.12 Based on the fact that an OLC with inhibitory activity on vascular Na-K-ATPase and therefore, vasoconstrictor activity, could be overproduced after brain injury, we hypothesized that the blockade of this substance with digoxin antibody would be beneficial in this circumstance.13 This study was conducted to determine whether an OLC is involved in BP changes of awake animals submitted to ICV hemorrhage. In addition, the effects of ICV infusion of digoxin antibody as a therapeutic approach were evaluated.
Received July 22, 2003. First decision August 5, 2003. Accepted August 27, 2003. From the Basic Research Laboratory, Nephrology (LIM-12), Medicine School, Sao Paulo University, Sao Paulo, Brazil. This work was supported by Fundac¸a˜o de Amparo a Pesquisa do
Estado de Sa˜o Paulo, FAPESP. Address correspondence and reprint requests to Dr. Varujan Dichtchekenian, Faculdade de Medicina da Universidade de Sao Paulo Av. Dr. Arnaldo, 455 3, andar-sala 3310 Cerqueira Ce´sar, Sao Paulo, Brasil CEP 01246 903; e-mail: [email protected]
0895-7061/03/$30.00 doi:10.1016/j.amjhyper.2003.08.001
Methods Animals Male Wistar rats weighing 250 to 350 g were used. Ad libitum diet containing 0.6 Na g/100 mL and tap water was given. The experimental protocol was approved by the animal studies ethical committee of Sao Paulo University Medical School. Surgical Implant of the Cannula A skin incision, under anesthesia with thiopental sodium (50 mg/kg intraperitoneally) was performed, and a stain-
© 2003 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.
AJH–December 2003–VOL. 16, NO. 12
DIGIBIND DECREASES BP IN CEREBRAL HEMORRHAGE
1063
Table 1. Tail– cuff BP, BP increase, cumulative sodium balance, and serum inhibitory activity on Na-KATPase Blood Pressure (mm Hg) D2 Blood Blood/ab CSF-like Control
117 125 128 100
⫾ ⫾ ⫾ ⫾
D4 6 3 6 1b
125 123 127 101
⫾ ⫾ ⫾ ⫾
D6 3 6 4 2
125 120 113 102
⫾ ⫾ ⫾ ⫾
D11 5 4 2 1
132 119 110 103
⫾ ⫾ ⫾ ⫾
a
2 4 2 3
⌬BP (mm Hg)
CSB (mEq Na/100g/6d)
IA% (%)
D11–D2
D6–D11
D11
14 ⫺7 ⫺19 3
⫾ ⫾ ⫾ ⫾
c
6 5 7 2
1.8 1.2 1.5 2.0
⫾ ⫾ ⫾ ⫾
0.1 0.2 0.4 0.5
19 ⫺13 ⫺72 ⫺19
⫾ ⫾ ⫾ ⫾
15d 25 7 3
a
P ⬍ .05 D2 v D11. b P ⬍ .05 control v blood, blood/ab, and CSF-like. c P ⬍ .05 blood v blood/ab, and CSF-like. d P ⬍ .05 blood v CSF-like. D ⫽ day; D2, D4 ⫽ before ICV infusions; D6, D11 ⫽ after ICV infusions; ⌬BP ⫽ D11–D2; CSB ⫽ cumulative sodium balance from D6 to D11; IA% ⫽ inhibitory activity of serum on Na-K-ATPase. Groups: Blood ⫽ ICV hemorrhage (n ⫽ 6); blood/ab ⫽ ICV hemorrhage/ ICV digoxin antibody (n ⫽ 5); CSF-like ⫽ ICV cerebrospinal-like solution (n ⫽ 6); Control ⫽ no ICV cannula or infusions (n ⫽ 5).
less steel cannula was introduced stereotaxically down to the right lateral ventricle. Brain histology was performed to confirm the site of ICV infusion. Animal Groups and ICV Infusions Group 1 animals were ICV infused with autologous venous blood, through an infusion pump at the rate of 50 L/12 min (n ⫽ 6). Group 2 animals were ICV infused with Fab fragments of digoxin antibody (DIGIBIND, Glaxo-Smith-Kline, Victoria, Australia; 10 mg/mL) just before ICV blood infusion at the rate of 10 L/2.4 min (n ⫽ 5). Group 3 animals were ICV infused with a cerebrospinal fluid (CSF)-like solution containing: Na⫹ 140 mEq/L, K⫹ 3 mEq/L, Ca2⫹ 1.3 mEq/L, Mg2⫹ 1 mEq/L, HCO3 25 mEq/L, and glucose 60 mg/dL, at a rate of 50 L/12 min (n ⫽ 6). Group 4 (control group) was anesthetized and sham operated on the same day of cannula introduction and ICV infusions (n ⫽ 5). Laboratory Measurements One week after cannula implant, the animals were placed in metabolic cages for an adaptation period of 3 days, and subsequently maintained for 11 days. The ICV infusions were performed on the fourth day under anesthesia (thiopental sodium 50 mg/kg intraperitoneally). Collections performed on the fifth day were discarded due to the influence of the anesthesia. Tail-cuff BP was measured on days 2, 4 (before), 6, and 11 (after) ICV infusions, six times on each day. Daily chow ingestion and urinary sodium excretion were measured to determine cumulative sodium balance. Urinary sodium concentration was measured with a flame photometer (CELM FC-280, Sao Paulo, Brazil). On the last day (day 11) before killing, blood samples were collected by cardiac puncture, under anesthesia, to measure the effects on Na-K-ATPase activity in renal outer medulla homogenate of normal rats. The [32 P]ATP hydrolysis method was used to determine enzyme activity.14
Na-K-ATPase Assay and Serum Inhibitory Activity on Normal Rat Renal Na-K-ATPase Activity Under anesthesia, normal rats were nephrectomized. Kidneys were sliced longitudinally, and outer medulla separated and homogenized for Na-K-ATPase assay. Four solutions were prepared to avoid influence of any other enzyme activity (solution A: 20 mmol/L KCl; solution B: 20 mmol/L KCl ⫹ 100 L of ouabain [0.018 g/mL; Sigma, St. Louis, MO]; solution C: no KCl ⫹ 100 L of ouabain; and solution D: 20 mmol/L KCl ⫹ 100 L of filtered animal serum). To estimate the effects of animal serum on Na-KATPase activity, comparison of maximal enzymatic activity (solution A) with the difference between solutions D and C were performed. Inhibition or stimulation of the enzyme was calculated. Statistical Analysis The analysis of variance test was used to compare independent groups. Blood pressure was analyzed with repeated measures (ANOVA). Spearman test was used for correlation. P ⬍ .05 was statistically significant. Results were expressed as mean ⫾ SEM.
Results Tail-cuff BP Table 1 shows tail-cuff BP. Between days 2 and 11 the following changes were observed: ICV blood infusion increased BP (P ⬍ .05), which could be prevented (P ⬎ .05) by digoxin antibody; CSF-like solution infusion decreased BP (P ⬍ .05) and control rats did not change BP (P ⬎ .05). Control animals presented lower BP before infusion (day 2, P ⬍ .05) compared to the other three groups. The BP difference (⌬BP) between day 11 and day 2 showed higher increase in ICV blood infused compared with ICV blood plus antibody and CSF-like rats (P ⬍ .05).
1064
DIGIBIND DECREASES BP IN CEREBRAL HEMORRHAGE
Cumulative Sodium Balance Table 1 shows cumulative sodium balance after ICV infusions in the four groups. No statistical difference was observed between groups (P ⬎ .05). Serum Inhibitory Activity on Na-K-ATPase Activity The percent inhibition (positive value) or stimulation (negative value) of rat serum on Na-K-ATPase activity of the outer medulla of normal rats is shown in Table 1. Serum of ICV blood rats showed Na-K-ATPase activity inhibition that was reversed by digoxin antibody to stimulation in ICV blood plus antibody rats. Serum of CSF-like and control animals showed stimulation of Na-K-ATPase activity. A significant difference between ICV blood and CSF-like animals was observed (P ⬍ .05). Correlation Between BP increase (⌬BP mm Hg) and serum inhibitory activity on Na-K-ATPase activity (IA%) A positive correlation between ⌬BP and IA% in ICV blood and ICV blood plus antibody groups, with R ⫽ 0.63 (P ⬍ .05) was observed. The ICV blood rats presented higher BP and IA% increase compared with ICV blood plus antibody rats.
AJH–December 2003–VOL. 16, NO. 12
in intracranial pressures to explain the BP increase. The type of ICV infused fluid and its effects on BP were analyzed and showed that blood, but not CSF-like solution, induced hypertension. Finally, according to Blaustein et al,9 endogenous OLC could inhibit vascular smooth muscle cell sodium pump increasing cytosolic sodium concentration and exchanging it for calcium. The high cytosolic calcium would increase vascular peripheral reactivity and BP. Digoxin antibody, used for digitalis intoxication treatment,13 was used based on its cross-reactivity with the endogenous hormone. The mechanism by which ICV infusion of digoxin antibody reduced BP is unknown. Hypothalamic or even vascular (the blood– brain barrier could be ruptured) action on the endogenous ouabain may explain these beneficial effects. In conclusion, cerebral hemorrhage increased BP through sodium pump inhibition. Digoxin antibody prevented BP increase.
Acknowledgments We thank the following people and institutions: 1) Dr. Antonio C Seguro and Dr. Lui Yu, Sao Paulo University, School of Medicine; 2) Hospital Israelita Albert Einstein; and 3) FAPESP, Fundac¸ a˜ o de Amparo a Pesquisa do Estado Sa˜ o Paulo.
Discussion An endogenous OLC with inhibitory activity on vascular Na-K-ATPase may be overproduced after brain injury, inducing an increase in BP. Therefore, we hypothesized that blockade of this substance with digoxin antibody may reduce BP after brain injury. Results confirmed this hypothesis. The ICV blood infusion increased BP and serum inhibitory Na-K-ATPase activity, which could be reversed with ICV digoxin antibody. The positive correlation between BP increase and Na-K-ATPase activity in ICV blood and ICV blood plus antibody confirmed these results. Available data in the literature indicate that cerebral manipulations induce, acutely, high intracranial pressure and BP, as seen in increased CSF sodium concentration and subarachnoid hemorrhage models.11,15 In this study, before ICV infusions (day 2), all three cannulated groups presented higher (P ⬍ .05) BP compared to the control rats. It is possible that the stress induced by cannulation could explain this higher baseline BP. On the other hand, ICV blood rats did not have an increase in BP (during the first 48 h). Instead, they have elevated BP 7 days after ICV infusion. Thus, it is unlikely that this late BP increase occurred due to experimental manipulations because control rats did not have an increase in BP and, on the contrary, CSF-like animals had decreased BP. Brain histology, performed 7 days after ICV infusions, did not show ventricle size increase, suggesting no later changes
References 1.
2. 3.
4.
5. 6.
7.
8.
9.
Donnan GA, Davis SM, Thrift A: The role of blood pressure lowering before and after stroke. Curr Opin Neurol 2003;16:81– 86. Labovitz DL, Sacco RL: Intracerebral hemorrhage: update. Curr Opin Neurol 2001;14:103–108. Perry HM Jr, Davis BR, Price TR, Applegate WB, Fields WS, Guralnik JM, Kuller l, Pressel S, Stamler J, Probstfield JL: Effect of treating isolated systolic hypertension on the risk of developing various types and subtypes of stroke: the Systolic Hypertension in the Elderly Program (SHEP). JAMA 2000;284:465–471. PROGRESS Collaborative Group: Randomised trial of a perindopril-based blood-pressure-lowering regimen among 6,105 individuals with previous stroke or transient ischaemic attack. Lancet 2001; 358:1033–1041. Loyke HF: The three phases of blood pressure in stroke. South Med J 1990;83:660 –663. Huang BS, Leenen FHH: Brain ouabain-like activity and the sympathoexitatory and pressor effects of central sodium in rats. Circ Res 1992;71:1059 –1066. Huang BS, Leenen FHH: Brain renin-angiotensin system and ouabain-induced sympathetic hyperactivity and hypertension in Wistar rats. Hypertension 1999;34:107–112. de Wardener HE, Mills IH, Clapham WF, Hayter CJ: Studies on the efferent mechanism of the sodium diuresis which follows the administration of intravenous saline in the dog. Clin Sci 1961;21:249 – 258. Blaustein MP, Juhaszova M, Golovina VA: The cellular mechanism of action of cardiotonic steroids: a new hypothesis. Clin Exp Hypertens 1998;20:691–703.
AJH–December 2003–VOL. 16, NO. 12
10. Wijdicks EFM, Vermeulen M, van Brummelen P: Digoxin-like immunoreactive substance in patients with aneurysmal subarachnoid hemorrhage. Br Med J 1987;294:729 –732. 11. Yamada K, Goto A, Nagoshi H, Hui C, Omata M: Role of brain ouabain like compound in central nervous system-mediated natriuresis in rats. Hypertension 1994;23:1027–1031. 12. Dichtchekenian V, Gisiger S, Quental I, Santos SR, Marcondes M, Heimann JC: Higher salt consumption, digoxin-like factor, and nifedipine response are associated with salt sensitivity in essential hypertension. Am J Hypertens 1992;5:707–712.
DIGIBIND DECREASES BP IN CEREBRAL HEMORRHAGE
1065
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