doi: 10.1161/01.STR.0000247868.97078.10
(Stroke. 2007;38:759.)
© 2007 American Heart Association, Inc.
Intracerebral Hemorrhage: Introduction |
Brain Injury After Intracerebral Hemorrhage
The Role of Thrombin and Iron
From the Department of Neurosurgery, University of Michigan, Ann Arbor.
Correspondence and reprint requests to Guohua Xi, MD, R5018 Biomedical Science Research Bldg, University of Michigan, 109 Zina Pitcher Pl, Ann Arbor, MI 48109-2200. E-mail guohuaxi{at}umich.edu
Abstract
Intracerebral hemorrhage (ICH) is a subtype of stroke with high morbidity and mortality. The mechanisms underlying ICH-induced brain injury have become better understood during the past decade. Experimental investigations have indicated that thrombin formation, red blood cell lysis, and iron toxicity play a major role in ICH-induced injury and that these mechanisms may provide new therapeutic targets. This article reviews the role of thrombin and iron in ICH-induced injury.
Key Words: brain edema • cerebral hemorrhage • iron • thrombin
Spontaneous intracerebral hemorrhage (ICH) often causes immediate death. If the patient survives from the ictus, the hematoma can lead to secondary brain injury and sometimes delayed fatality. Recent studies have helped to elucidate the mechanisms that trigger pathophysiological changes in and around the hematoma. In particular, they have focused attention on the role of thrombin and iron, released upon red blood cell (RBC) lysis, as 2 major factors causing brain injury after ICH.
Thrombin and Brain Injury After ICH
Thrombin-Induced Brain Injury
Thrombin is a serine protease and an essential component in the coagulation cascade. It is produced immediately in the brain after an ICH to stop the bleeding. However, direct infusion of large doses (5 U) of thrombin into brain causes inflammatory cell infiltration, brain edema formation, and neuronal death.1,2 We have demonstrated that thrombin is responsible for early brain edema formation after ICH and that such edema results partly from a direct opening of the blood-brain barrier.3 Because thrombin can be detrimental at high concentrations and protective at low concentrations, it is important to know the concentrations of thrombin that may occur in the brain after ICH. The concentration of prothrombin in the plasma is high enough (1 to 5 µmol/L) to produce a substantial amount of thrombin in the brain parenchyma after a hemorrhage. Thus, 1 mL of whole blood can produce 
260 to 360 U of thrombin, and a 50-µL clot could be expected to produce up to 15 U of thrombin. In the rat, the brain edema produced by intracerebral hematoma can be significantly inhibited by thrombin inhibitors, and an intracerebral infusion of 5 U of thrombin causes a degree of edema similar to that of a 50-µL clot.4,5 Therefore, this dose of thrombin was used in our previous experiments.6
To examine whether delayed and systemic administration of a thrombin inhibitor could reduce ICH-induced injury, experiments were performed with argatroban. Intracerebral infusion of blood caused a marked increase in perihematomal water content. Intracerebral injection of argatroban 3 hours after ICH caused a significant reduction in edema measured at 48 hours. The systemic administration of high-dose argatroban (0.9 mg/h) starting 6 hours after ICH also significantly reduced edema.7
Advances have been made in understanding the manner in which intracerebral thrombin may have adverse effects on the brain. Thrombin at high concentrations kills neurons and astrocytes in vitro.1 Thus, to assess the direct toxic effects of thrombin on brain cells, mixed rat neuron/astrocyte cultures were exposed to different doses of thrombin (1, 2, 5, 10, 20, 50, or 100 U/mL) for 24 hours, and media lactate dehydrogenase concentrations were determined as an indicator of cell viability. Low doses of thrombin (1 and 2 U/mL) did not induce cell death. However, doses >5 U/mL resulted in dose-dependent lactate dehydrogenase release.8
Thrombin-induced brain injury may be mediated by the complement cascade.9 Intracerebral infusion of thrombin in rat resulted in a 7-fold increase in complement C9 and deposition of complement C9 on neuronal membranes. Clusterin, an inhibitor of the membrane attack complex formation, was also upregulated by the thrombin and found in neurons. The effects of coagulation cascade on complement activation are not well studied. However, studies suggest that there is a very close relationship between thrombin and complement. For example, thrombin-cleaved C3a-like fragments are chemotactic for leukocytes and induce enzyme release from neutrophils.10
Tumor necrosis factor-
(TNF-) is one of the major proinflammatory cytokines, and it may contribute to brain injury after ICH. TNF- levels in the brain are increased after intracerebral infusion of thrombin and ICH.11 ICH-induced brain edema was less in TNF- knockout mice than in wild-type mice (Figure 1). In addition, thrombin activates matrix metalloproteinase-2 in endothelial cells.12 Matrix metalloproteinases are members of a family of zinc-dependent proteases that can degrade extracellular matrix and cause blood-brain barrier disruption.
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Intracerebral infusion of thrombin does not cause ischemic brain damage. Thus, after infusion of 10 U of thrombin in the rat, cerebral blood flow declined over the first hour, rose to baseline or above by 2 hours, and returned to baseline by 24 hours. The lowest blood flow, 45±7 mL/100 g per minute, was recorded in the ipsilateral hemisphere at 1 hour, and this is well above the values expected to cause ischemic damage.3
Thrombin-Induced Neuroprotection
Thrombin can also have beneficial effects in ICH. Thus, it is involved in stopping the initial bleed and preventing hematoma enlargement that occurs in a percentage of ICH patients over the first day. This latter observation forms the basis of current factor VIIa trials.13 In addition, many studies indicate that although high concentrations of thrombin produce deleterious effects, brain exposure to low concentrations can induce protective effects. Thrombin preconditioning reduces brain injury in models of ICH and focal cerebral ischemia.1 Although the precise mechanisms of thrombin-induced brain tolerance to hemorrhagic and ischemic stroke are not known, activation of thrombin receptors, upregulation of iron handling proteins, and heat shock proteins in the brain may be associated with the induced tolerance.1,14
RBC Lysis, Iron, and ICH-Induced Brain Damage
RBC Lysis and Brain Edema Formation
Lysis of RBCs occurs several days after ICH, which results from either depletion of intracellular energy reserves or formation of membrane attack complex after activation of the complement system, or both.15,16 RBC lysis contributes to edema formation after ICH. A clinical study of edema and ICH indicates that delayed brain edema is related to significant midline shift after ICH in humans.17 This delayed brain edema (in the second or third week after onset in humans) is probably attributable to hemoglobin and its degradation products. Another recent study in ICH patients also suggests that delayed brain edema after ICH may result from hematoma lysis.18 A total of 17 spontaneous ICH patients were chosen in that study. All patients had the first CT scan within 5 hours of symptom onset and then underwent second, third, and fourth CT scans at 1, 3, and 10 days later. That study found that hematoma size was reduced significantly at day 10 as a result of clot lysis. However, edema volume increased gradually over that time.
In animal studies, an intracerebral infusion of lysed RBCs but not packed RBCs resulted in marked brain edema formation and DNA injury within 24 hours.19,20 However, the infusion of packed RBCs caused edema and neurological deficits several days later, suggesting that RBCs are associated with delayed brain edema formation.6,19
Whether or not ischemia contributes to perihematomal edema development is still controversial. An intracerebral infusion of RBC hemolysate induced marked edema, but cerebral blood flow in the vicinity remained near normal. It appears that the edema formation is related to marked blood-brain barrier disruption from the toxic effect of lysed RBCs rather than from ischemia.21
Heme is degraded by heme oxygenase (HO) in the brain into iron, carbon monoxide, and biliverdin. An intracerebral infusion of hemoglobin or its degradation products caused brain edema within 24 hours. Our recent studies have shown that HO-1 protein levels are increased after ICH.22 The biological significance of HO-1 upregulation is still uncertain, but tin-mesoporphyrin, a HO inhibitor, attenuates perihematomal edema in a pig ICH model and reduces neuronal loss in a rabbit ICH model.23,24
Intracerebral infusion of iron causes brain edema, and iron can exacerbate thrombin-induced brain edema.25,26 When RBCs start to lyse, hemoglobin degradation can result in iron release. The iron concentration could reach as high as 10 mmol/L, which causes marked brain edema.25 Our recent study found that ferrous iron at low concentrations (for example, 0.2 mmol/L) can also induce brain damage.27 Deferoxamine, an iron chelator, reduces hematoma- and hemoglobin-induced edema, suggesting that iron plays an important role in edema formation after ICH.28
Iron Overload, Brain Atrophy, and Neurological Deficits After ICH
After erythrocyte lysis, iron concentrations in the brain reach very high levels. We found a 3-fold increase of brain nonheme iron after ICH in rats, and this level remained high for at least 1 month.22 Iron-induced brain damage may result from oxidative stress.29 Antioxidants block neuronal toxicity induced by hemoglobin.30,31
Clinical and experimental studies have demonstrated that brain atrophy occurs after ICH.32,33 The underlying cause(s) of this atrophy is, however, unknown. In a recent study, we demonstrated that brain atrophy developed gradually and peaked between 1 and 2 months after ICH in the rat33 (Figure 2). Brain atrophy was associated with prolonged neurological deficits. Deferoxamine, an iron chelator, reduced brain atrophy (Figure 3), reduced brain ferritin immunoreactivity, and improved neurological deficits after rat ICH.
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The Relationship Among Brain Edema, Cell Death, and Neurological Deficits
A rat ICH model involving infusion of autologous blood into the caudate has been used extensively to study mechanisms of brain edema formation.16,19,34–36 It has, however, been difficult to obtain other quantifiable markers of brain injury because neuronal injury appears to be diffuse (ie, there is no defined infarct), and only a small cavity is found after the clot is absorbed. However, our recent study showed that there is a correlation between ICH-induced neurological deficits and brain edema.6 Although it is not clear whether edema exaggerates cell death, we have demonstrated that deferoxamine reduces brain edema, brain atrophy, and neurological deficits in a rat model of ICH.28,33
Summary
Thrombin causes brain damage at high concentrations and induces neuroprotection at low concentrations. Modulating brain thrombin activity may establish novel therapeutic strategies for ICH, particularly if methods can be devised to elicit only beneficial effects. Iron is a major factor causing ICH-induced brain injury. Deferoxamine, an iron chelator, reduces ICH-induced brain edema, behavioral deficits, and atrophy, suggesting that it may be a useful therapy for ICH patients.
Acknowledgments
Sources of Funding
This study was supported by grants NS-17760, NS-39866, and NS-47245 from the National Institutes of Health and Scientist Development Grant 0435354Z from the American Heart Association.
Disclosures
None.
Footnotes
Presented at the 25th Princeton Conference, Session VII: Intracerebral Hemorrhage, held in Portland, Oregon, May 19–20, 2006.
Received June 16, 2006; revision received August 21, 2006; accepted August 31, 2006.
References
1. Xi G, Reiser G, Keep RF. The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury: deleterious or protective? J Neurochem. 2003; 84: 3–9.[CrossRef][Medline] [Order article via Infotrieve]
2. Xi G, Keep R, Hoff J. Mechanisms of brain injury after intracerebral hemorrhage. Lancet Neurol. 2006; 5: 53–63.[CrossRef][Medline] [Order article via Infotrieve]
3. Lee KR, Kawai N, Kim S, Sagher O, Hoff JT. Mechanisms of edema formation after intracerebral hemorrhage: effects of thrombin on cerebral blood flow, blood-brain barrier permeability, and cell survival in a rat model. J Neurosurg. 1997; 86: 272–278.[Medline] [Order article via Infotrieve]
4. Lee KR, Colon GP, Betz AL, Keep RF, Kim S, Hoff JT. Edema from intracerebral hemorrhage: the role of thrombin. J Neurosurg. 1996; 84: 91–96.[Medline] [Order article via Infotrieve]
5. Xi G, Wagner KR, Keep RF, Hua Y, de Courten-Myers GM, Broderick JP, Brott TG, Hoff JT. The role of blood clot formation on early edema development following experimental intracerebral hemorrhage. Stroke. 1998; 29: 2580–2586.
6. Hua Y, Schallert T, Keep RF, Wu J, Hoff JT, Xi G. Behavioral tests after intracerebral hemorrhage in the rat. Stroke. 2002; 33: 2478–2484.
7. Kitaoka T, Hua Y, Xi G, Hoff JT, Keep RF. Delayed argatroban treatment reduces edema in a rat model of intracerebral hemorrhage. Stroke. 2002; 33: 3012–3018.
8. Jiang Y, Wu J, Hua Y, Keep RF, Xiang J, Hoff JT, Xi G. Thrombin-receptor activation and thrombin-induced brain tolerance. J Cereb Blood Flow Metab. 2002; 22: 404–410.[Medline] [Order article via Infotrieve]
9. Gong Y, Xi G, Keep R, Hoff J, Hua Y. Complement inhibition attenuates brain edema and neurological deficits induced by thrombin. Acta Neurochir (Wien). 2005; 95 (suppl): 389–392.
10. Hugli TE. Complement factors and inflammation: effects of thrombin on components of c3 and c5. In: Lundblad RL, Fenton JW, Mann KG, eds. Chemistry and Biology of Thrombin. Ann Arbor, Mich: Ann Arbor Science Publishers; 1977: 345–360.
11. Hua Y, Wu J, Keep R, Nakamura T, Hoff J, Xi G. Tumor necrosis factor-alpha increases in the brain following intracerebral hemorrhage and thrombin stimulation. Neurosurgery. 2006; 58: 542–550.[Medline] [Order article via Infotrieve]
12. Nguyen M, Arkell J, Jackson CJ. Thrombin rapidly and efficiently activates gelatinase a in human microvascular endothelial cells via a mechanism independent of active mt1 matrix metalloproteinase. Lab Invest. 1999; 79: 467–475.[Medline] [Order article via Infotrieve]
13. Mayer SA, Brun NC, Begtrup K, Broderick J, Davis S, Diringer MN, Skolnick BE, Steiner T. Recombinant activated factor VII for acute intracerebral hemorrhage. N Engl J Med. 2005; 352: 777–785.
14. Hua Y, Keep RF, Hoff JT, Xi G. Thrombin preconditioning attenuates brain edema induced by erythrocytes and iron. J Cereb Blood Flow Metab. 2003; 23: 1448–1454.[CrossRef][Medline] [Order article via Infotrieve]
15. Kase CS, Caplan LR. Intracerebral Hemorrhage. Boston, Mass: Butterworth-Heinemann; 1994.
16. Hua Y, Xi G, Keep RF, Hoff JT. Complement activation in the brain after experimental intracerebral hemorrhage. J Neurosurg. 2000; 92: 1016–1022.[Medline] [Order article via Infotrieve]
17. Zazulia AR, Diringer MN, Derdeyn CP, Powers WJ. Progression of mass effect after intracerebral hemorrhage. Stroke. 1999; 30: 1167–1173.
18. Wu G, Xi G, Huang F. Spontaneous intracerebral hemorrhage in humans: hematoma enlargement, clot lysis, and brain edema. Acta Neurochir (Wien). 2006; 96 (suppl): 78–80.
19. Xi G, Keep RF, Hoff JT. Erythrocytes and delayed brain edema formation following intracerebral hemorrhage in rats. J Neurosurg. 1998; 89: 991–996.[Medline] [Order article via Infotrieve]
20. Wu J, Hua Y, Keep RF, Schallert T, Hoff JT, Xi G. Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage. Brain Res. 2002; 953: 45–52.[CrossRef][Medline] [Order article via Infotrieve]
21. Xi G, Hua Y, Bhasin RR, Ennis SR, Keep RF, Hoff JT. Mechanisms of edema formation after intracerebral hemorrhage: effects of extravasated red blood cells on blood flow and blood-brain barrier integrity. Stroke. 2001; 32: 2932–2938.
22. Wu J, Hua Y, Keep RF, Nakamura T, Hoff JT, Xi G. Iron and iron-handling proteins in the brain after intracerebral hemorrhage. Stroke. 2003; 34: 2964–2969.
23. Wagner KR, Hua Y, de Courten-Myers GM, Broderick JP, Nishimura RN, Lu SY, Dwyer BE. Tin-mesoporphyrin, a potent heme oxygenase inhibitor, for treatment of intracerebral hemorrhage: in vivo and in vitro studies. Cell Mol Biol. 2000; 46: 597–608.[Medline] [Order article via Infotrieve]
24. Koeppen AH, Dickson AC, Smith J. Heme oxygenase in experimental intracerebral hemorrhage: the benefit of tin-mesoporphyrin. J Neuropathol Exp Neurol. 2004; 63: 587–597.[Medline] [Order article via Infotrieve]
25. Huang F, Xi G, Keep RF, Hua Y, Nemoianu A, Hoff JT. Brain edema after experimental intracerebral hemorrhage: role of hemoglobin degradation products. J Neurosurg. 2002; 96: 287–293.[Medline] [Order article via Infotrieve]
26. Nakamura T, Xi G, Park JW, Hua Y, Hoff JT, Keep RF. Holo-transferrin and thrombin can interact to cause brain damage. Stroke. 2005; 36: 348–352.
27. Yang S, Hua Y, Nakamura T, Keep RF, Xi G. Up-regulation of brain ceruloplasmin in thrombin preconditioning. Acta Neurochir Suppl. 2006; 96: 203–206.[Medline] [Order article via Infotrieve]
28. Nakamura T, Keep R, Hua Y, Schallert T, Hoff J, Xi G. Deferoxamine-induced attenuation of brain edema and neurological deficits in a rat model of intracerebral hemorrhage. J Neurosurg. 2004; 100: 672–678.[CrossRef][Medline] [Order article via Infotrieve]
29. Wagner KR, Sharp FR, Ardizzone TD, Lu A, Clark JF. Heme and iron metabolism: role in cerebral hemorrhage. J Cereb Blood Flow Metab. 2003; 23: 629–652.[CrossRef][Medline] [Order article via Infotrieve]
30. Regan RF, Panter SS. Neurotoxicity of hemoglobin in cortical cell culture. Neurosci Lett. 1993; 153: 219–222.[CrossRef][Medline] [Order article via Infotrieve]
31. Wang X, Mori T, Sumii T, Lo EH. Hemoglobin-induced cytotoxicity in rat cerebral cortical neurons: caspase activation and oxidative stress. Stroke. 2002; 33: 1882–1888.
32. Felberg RA, Grotta JC, Shirzadi AL, Strong R, Narayana P, Hill-Felberg SJ, Aronowski J. Cell death in experimental intracerebral hemorrhage: the "black hole" model of hemorrhagic damage. Ann Neurol. 2002; 51: 517–524.[CrossRef][Medline] [Order article via Infotrieve]
33. Hua Y, Nakamura T, Keep R, Wu J, Schallert T, Hoff J, Xi G. Long-term effects of experimental intracerebral hemorrhage: the role of iron. J Neurosurg. 2006; 104: 305–312.[Medline] [Order article via Infotrieve]
34. Yang GY, Betz AL, Chenevert TL, Brunberg JA, Hoff JT. Experimental intracerebral hemorrhage: relationship between brain edema, blood flow, and blood-brain barrier permeability in rats. J Neurosurg. 1994; 81: 93–102.[Medline] [Order article via Infotrieve]
35. Xi G, Hua Y, Keep RF, Younger JG, Hoff JT. Systemic complement depletion diminishes perihematomal brain edema. Stroke. 2001; 32: 162–167.
36. Hua Y, Xi G, Keep RF, Wu J, Jiang Y, Hoff JT. Plasminogen activator inhibitor-1 induction after experimental intracerebral hemorrhage. J Cereb Blood Flow Metab. 2002; 22: 55–61.[CrossRef][Medline] [Order article via Infotrieve]
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