**1. Introduction**

Intracerebral hemorrhage (ICH) is a type of acute stroke characterized by extravasation of blood into brain parenchyma and formation of hematoma, leading to edema and tissue damage in the brain. During ICH, rapid accumulation of blood within brain parenchyma leads to disruption of normal anatomy and increased local pressure. Depending on the dynamic of hematoma expansion (growth), the primary damage occurs within minutes to hours from the onset of bleeding and is primarily the result of mechanical damage associated with the mass effect, which compress adjacent tissues, thus destroying them. The 'mass effect' is an important factor in the pathogenic events in ICH. But it may be difficult to predict and manage this effect directly by drug therapies [1-2].

Once present, ICH causes both primary and secondary injury. The primary insult is due to disruption of adjacent tissue and mass effect. Secondary injury occurs with the development of edema, free radical formation, inflammation, and direct cellular toxicity due to the deposited hematoma and subsequent degradation byproducts [3].

### **2. Body**

After arteriolar rupture and parenchymal hemorrhage in the brain, a combination of local compression, cytotoxic injury, inflammation, and surrounding edema ensues. Many patients with ICH deteriorate progressively with no sign of hematoma expansion, suggesting that secondary damage following ICH plays a critical role in neurological deterioration. Secondary damage is, for the most part, attributable to the presence of intraparenchymal blood and may be dependent on the initial hematoma volume, patients age, or ventricu‐

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lar volume. Several lines of evidence show that secondary damage involves blood constit‐ uents such as thrombin and hemoglobin as well as its degradation products, which exert biological actions or toxic influences on brain cells. These events, primarily resulting from blood extravasation, which subsequently activates cytotoxic, oxidative and inflammatory pathways, also triggers secondary reactions in the brain parenchyma including recruit‐ ment of additional proteases, cerebral edema, and cellular apoptosis, ultimately leading to blood–brain barrier disruption and massive brain cell death. The toxic effects of extravasat‐ ed blood result mainly from blood components, including red blood cells (RBCs), plasma proteins, coagulation factors, inflammatory mediators , complement components and immunoglobulins. After ICH, the extravasated blood components (primarily erythrocytes and plasma proteins) and the damage-associated molecular patterns, impose a strong cytotoxic, pro-oxidative, and proinflammatory insult toward adjacent viable brain cells and could be seen as early as minutes after onset of ICH [1-3].

**3.1. Thrombin [1-2]**

tration of the thrombin inhibitor hirudin.

the late stage may result in edema formation and be toxic.

Thrombin, a serine protease produced rapidly after ICH onset, plays a pivotal role in the blood coagulation cascade. In response to bleeding, a complex series of clotting-factor interactions leads to its conversion by thromboplastin to thrombin, which transforms fibrinogen in plasmainto fibrin, as well as catalyzing many other coagulation-related reactions. As part of its activity in the coagulation cascade, thrombin also promotes platelet activation and aggre‐ gation via activation of protease-activated receptors on the cell membrane of the platelet. The primary purpose of thrombin is to stop bleeding as soon as possible and prevent hematoma expansion. Besides its physiological role, substantial lines of evidence indicate that thrombin participates in various pathological conditions in the brain, which contributes to edema formation and blood–brain barrier damage in early brain injury, and activates the cytotoxic, excitotoxic and inflammatory pathways that are involved in secondary injury following ICH.

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27

In the case of ICH, a large amount of blood-derived thrombin invades the brain tissue and exerts biological actions through its proteolytic activity. The substrates for thrombin include proteinase-activated receptors that transduce intracellular signals via trimeric G proteins.The role of thrombin in ICH pathogenesis was first suggested by its possible involvement in edema formation. That is, injection of whole blood into the striatum of rats induced edema, which was prevented by addition of hirudin, a thrombin inhibitor. Edema induced by autologous blood injection into the striatum was attenuated also by argatroban, another inhibitor of thrombin, even when the drug was systemically administered from 6 h after blood injection. ICH-associated edema results from disruption of the blood–brain barrier and death of brain parenchymal cells, both of which may be induced by thrombin.Application of thrombin to rat corticostriatal cultures induced delayed neuron death in the cortical region and shrinkage of the striatal region. Various pharmacological examinations revealed distinct properties of the mechanisms of injury between the cerebral cortex and the striatum. For example, extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), three major members of mitogen-activated protein kinase (MAPK) family, all contributed to thrombin-induced injury of the striatum, whereas ERK, but not p38 or JNK, was involved in cortical injury. In addition, depletion of microglia from slice cultures rescued striatal tissue, but not cortical cells, from thrombin-induced injury, suggesting that microglia participate only in striatal tissue injury. Involvement of MAPKs and activated microglia in striatal tissue injury was confirmed by an in vivo study where thrombin was directly injected into the striatum of adult rats. On the other hand, plasminogen was found to cooperate with thrombin in inducing cortical injury but not striatal injury.Thrombin-mediated cellular injury may also be mediated by activation of matrix metalloproteinase (MMP)-9. That is, concurrent application of MMP-9 exacerbates cytotoxicity of thrombin in neurons in primary culture, and thrombin cytotoxicity is partially attenuated by MMP inhibitors. Moreover, brain damage induced by autologous blood injection is synergistically attenuated by deletion of the gene encoding MMP-9 and adminis‐

So, Thrombin can be a double-edged sword, It should be protective during the early stage in ICH to rapidly stop bleeding and prevent hematoma enlargement , whereas its augment in

In addition to the hematoma, the associated edema may also contribute to the initial neurological deficit, subsequent decline, or death. The edema related to ICH has been cited as a reason for neurological deterioration after the first 24 to 48 h from the onset of symptoms, and it has, to a lesser degree, also been implicated with deterioration as late as 3 weeks. The edema has been demonstrated to be predominately vasogenic with a cytotoxic component. The vasogenic edema is a consequence of blood brain barrier (BBB) break‐ down. In the normal brain, the BBB prevents the flow of water into the brain due to hydrostatic pressure gradients. However, when the BBB is disrupted as occurs in ICH, the imbalance in hydrostatic forces result in the entry of an exudative proteinaceous fluid onto the brain parenchyma. The disruption in the BBB is likely a consequence of an inflammato‐ ry cascade with resultant expression of specific cytokines and other markers of inflamma‐ tion. The presence of red blood cells and their subsequent lysis and release of oxyhemoglobin may contribute to the leakage of the BBB. The hemorrhage itself also induces the production of thrombin and the overexpression of matrix metalloproteinases. Throm‐ bin has been demonstrated to be an important factor in the modulation of BBB break‐ down. Thrombin may be a major mediator of ICH-induced tumor necrosis factor-α production and an increase of perihematomal tumor necrosis factor-α levels contributes to brain edema formation after ICH. Matrix metalloproteinases also promote BBB disruption and have been associated with increased edema volume via extracellular matrix proteoly‐ sis, basal lamina destruction, and the degradation of c-fibronectin [4].

#### **3. Blood plasma components/products**

At early stage following ICH, the toxicity of extravasated blood plasma components including blood derived coagulation factors, complement components, immunoglobulins, and other bioactive molecules are proposed to act as contributors to ICH-affected tissue damage.

#### **3.1. Thrombin [1-2]**

lar volume. Several lines of evidence show that secondary damage involves blood constit‐ uents such as thrombin and hemoglobin as well as its degradation products, which exert biological actions or toxic influences on brain cells. These events, primarily resulting from blood extravasation, which subsequently activates cytotoxic, oxidative and inflammatory pathways, also triggers secondary reactions in the brain parenchyma including recruit‐ ment of additional proteases, cerebral edema, and cellular apoptosis, ultimately leading to blood–brain barrier disruption and massive brain cell death. The toxic effects of extravasat‐ ed blood result mainly from blood components, including red blood cells (RBCs), plasma proteins, coagulation factors, inflammatory mediators , complement components and immunoglobulins. After ICH, the extravasated blood components (primarily erythrocytes and plasma proteins) and the damage-associated molecular patterns, impose a strong cytotoxic, pro-oxidative, and proinflammatory insult toward adjacent viable brain cells and

In addition to the hematoma, the associated edema may also contribute to the initial neurological deficit, subsequent decline, or death. The edema related to ICH has been cited as a reason for neurological deterioration after the first 24 to 48 h from the onset of symptoms, and it has, to a lesser degree, also been implicated with deterioration as late as 3 weeks. The edema has been demonstrated to be predominately vasogenic with a cytotoxic component. The vasogenic edema is a consequence of blood brain barrier (BBB) break‐ down. In the normal brain, the BBB prevents the flow of water into the brain due to hydrostatic pressure gradients. However, when the BBB is disrupted as occurs in ICH, the imbalance in hydrostatic forces result in the entry of an exudative proteinaceous fluid onto the brain parenchyma. The disruption in the BBB is likely a consequence of an inflammato‐ ry cascade with resultant expression of specific cytokines and other markers of inflamma‐ tion. The presence of red blood cells and their subsequent lysis and release of oxyhemoglobin may contribute to the leakage of the BBB. The hemorrhage itself also induces the production of thrombin and the overexpression of matrix metalloproteinases. Throm‐ bin has been demonstrated to be an important factor in the modulation of BBB break‐ down. Thrombin may be a major mediator of ICH-induced tumor necrosis factor-α production and an increase of perihematomal tumor necrosis factor-α levels contributes to brain edema formation after ICH. Matrix metalloproteinases also promote BBB disruption and have been associated with increased edema volume via extracellular matrix proteoly‐

could be seen as early as minutes after onset of ICH [1-3].

26 Intracerebral Hemorrhage

sis, basal lamina destruction, and the degradation of c-fibronectin [4].

At early stage following ICH, the toxicity of extravasated blood plasma components including blood derived coagulation factors, complement components, immunoglobulins, and other bioactive molecules are proposed to act as contributors to ICH-affected tissue damage.

**3. Blood plasma components/products**

Thrombin, a serine protease produced rapidly after ICH onset, plays a pivotal role in the blood coagulation cascade. In response to bleeding, a complex series of clotting-factor interactions leads to its conversion by thromboplastin to thrombin, which transforms fibrinogen in plasmainto fibrin, as well as catalyzing many other coagulation-related reactions. As part of its activity in the coagulation cascade, thrombin also promotes platelet activation and aggre‐ gation via activation of protease-activated receptors on the cell membrane of the platelet. The primary purpose of thrombin is to stop bleeding as soon as possible and prevent hematoma expansion. Besides its physiological role, substantial lines of evidence indicate that thrombin participates in various pathological conditions in the brain, which contributes to edema formation and blood–brain barrier damage in early brain injury, and activates the cytotoxic, excitotoxic and inflammatory pathways that are involved in secondary injury following ICH.

In the case of ICH, a large amount of blood-derived thrombin invades the brain tissue and exerts biological actions through its proteolytic activity. The substrates for thrombin include proteinase-activated receptors that transduce intracellular signals via trimeric G proteins.The role of thrombin in ICH pathogenesis was first suggested by its possible involvement in edema formation. That is, injection of whole blood into the striatum of rats induced edema, which was prevented by addition of hirudin, a thrombin inhibitor. Edema induced by autologous blood injection into the striatum was attenuated also by argatroban, another inhibitor of thrombin, even when the drug was systemically administered from 6 h after blood injection. ICH-associated edema results from disruption of the blood–brain barrier and death of brain parenchymal cells, both of which may be induced by thrombin.Application of thrombin to rat corticostriatal cultures induced delayed neuron death in the cortical region and shrinkage of the striatal region. Various pharmacological examinations revealed distinct properties of the mechanisms of injury between the cerebral cortex and the striatum. For example, extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), three major members of mitogen-activated protein kinase (MAPK) family, all contributed to thrombin-induced injury of the striatum, whereas ERK, but not p38 or JNK, was involved in cortical injury. In addition, depletion of microglia from slice cultures rescued striatal tissue, but not cortical cells, from thrombin-induced injury, suggesting that microglia participate only in striatal tissue injury. Involvement of MAPKs and activated microglia in striatal tissue injury was confirmed by an in vivo study where thrombin was directly injected into the striatum of adult rats. On the other hand, plasminogen was found to cooperate with thrombin in inducing cortical injury but not striatal injury.Thrombin-mediated cellular injury may also be mediated by activation of matrix metalloproteinase (MMP)-9. That is, concurrent application of MMP-9 exacerbates cytotoxicity of thrombin in neurons in primary culture, and thrombin cytotoxicity is partially attenuated by MMP inhibitors. Moreover, brain damage induced by autologous blood injection is synergistically attenuated by deletion of the gene encoding MMP-9 and adminis‐ tration of the thrombin inhibitor hirudin.

So, Thrombin can be a double-edged sword, It should be protective during the early stage in ICH to rapidly stop bleeding and prevent hematoma enlargement , whereas its augment in the late stage may result in edema formation and be toxic.

#### **3.2. Haptoglobin (Hp) and Hemopexin(Hx) [1-2]**

Haptoglobin and Hemopexin Acts to Combat Hb/Heme Toxicity After ICH.

Haptoglobin (Hp), an acute phase protein, is an abundant blood plasma component that is normally synthesized and released into blood circulation primarily by hepatocytes. The primary function of Hp in blood is to bind and neutralize nephrotoxic free Hb in case of intravascular hemolysis.

**4. Red blood cells degradation products [1-2, 5-6]**

edema and the secondary brain damage after ICH.

chemicals capable of causing death to many brain cells.

**4.1. Hemoglobin (Hb) and Heme and heme oxygenase-1 (HO-1) [1-2, 5-6]**

Aside from proteases and plasma products , important blood constituents contributing to ICH pathogenesis are erythrocyte and its degradation products. Following ICH, a large number of red blood cells penetrate into brain parenchyma. Hemolysis does not occur promptly after ICH, but rather, proceeds slowly, taking several days to weeks. Packed RBCs do not cause acute edema development after infusion into the basal ganglia or frontal white matter. However, they do cause delayed edema that appears to be related to release of hemoglobin. Usually, most RBCs start to lyse several days after ICH, but RBC lysis can occur very early.A cascade of events triggered by erythrocyte lysis is critical for the delayed development of

The Pathogenesis of Edema and Secondary Insults after ICH

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29

Red blood cell lysis lead to release of cytotoxic hemoglobin (Hb) with further deterioration of the pathological status quo . Hb and its degradation products, heme and iron, directly compromise the well-being of neighboring brain cells. Hb and heme are potent cytotoxic

Hemoglobin(Hb) is a major component of blood and a potent mediator of oxidative stress after ICH. After a cerebral hemorrhage, large numbers of hemoglobin-containing red blood cells are released into the brain's parenchyma and/or subarachnoid space. Hemoglobin is released from lysed red blood cells, heme is liberated from hemoglobin, and hemoglobin as well as heme is taken up by brain parenchymal cells such as microglia and neurons. Prominently, the mechanism of hemoglobin toxicity is via generating free radicals (mainly through Fenton-type mechanism) and massive oxidative damage to proteins, nucleic acids, carbohydrates, and lipids. Heme itself or its degradation products may contribute to formation of brain edema and secondary brain injury after hemorrhagic insults. Hemin, the oxidative form of heme, plays a critical role in Hb-induced brain injury following ICH. Hemin exerts its neurotoxic effects via release of excessive iron, depletion of glutathione and production of free radicals.

Breakdown of the heme moieties of hemoglobin is catalyzed by heme oxygenase-1 (HO-1) into iron, carbon monoxide, and biliverdin, the latter two of which are thought to mediate the antiinflammatory and antioxidant actions. HO-1, the rate-limiting enzyme for heme catabolism and iron production, after induction of ICH by collagenase or injection of autologous blood, robust expression of HO-1 is induced predominantly in non-neural cells such as microglia/

Under normal conditions, HO-1 is expressed at a very low level, but it is rapidly induced by hemoglobin, heme, and various oxidants. HO-1 has a convincing role in the regulation of intracellular iron. HO-1increased heme catabolism, so it plays a protective role against oxidative injuries in the vascular system.HO-1 induction protects astrocytes from the oxidative toxicity of hemoglobin and heme. HO-1 has a protective role as an intrinsic factor against oxidative stress and the protection depends on the degree of oxidative stress by generating antioxidant bilirubin and vasodilating carbon monoxide. Deficiency of HO-1 in humans and

macrophages and endothelial cells. The role of HO-1 following ICH is controversial.

Under normal circumstances, Hp represents an effective mechanism by which our body is protected from Hb toxicity. However, because Hp synthesis is not increased by low Hp levels and Hp is not recycled by macrophages, it may take 5 to 7 days for the Hp level to recover if completely sequestered by Hb. Thus, massive hemolysis may lead to persistent hypohapto‐ globinemia. Interestingly, Recently study demonstrated that Hp is also produced locally in rat brain after ICH and its expression is significantly increased around the hematoma within hours from the onset of ICH.

The brain-derived Hp appeared to be synthesized and released by oligodendrocytes. Because oligodendroglia are abundant in white matter and are present throughout the gray matter, local production of Hp by these cells likely represents an important endogenous mechanism protecting brain against the extravascular Hb toxicity. Indirect support for such a claim includes (1) primary oligodendrocytes protect neurons in culture from Hb toxicity via Hp release; (2) animals made hypohaptoglobinemic with repetitive Hb administration, before ICH, experience more extensive brain damage; (3) mice genetically engineered to overexpress Hp are less susceptible to ICH injury; and (4) Hp-deficient mice are more vulnerable to ICH injury. In the context of therapeutic relevance, there have been determined that pharmacolog‐ ical intervention with sulforaphane, a naturally occurring agent that acts as NF-E2–related factor-2 (Nrf2) transcription factor activator, increases Hp in blood plasma and brain, and notably reduces brain damage in animal models of ICH. The relevance of Hp genotype as a factor modifying the outcome after ICH has not been studied to date. It could also be relevant for this review to indicate that in addition to the Hp-Hb/CD163 scavenging system, an independent system exists to help remove Hb breakdown products, heme, and iron.

Hemopexin (Hx) is a blood plasma glycoprotein synthesized primarily by hepatocytes. Hx has been shown to bind to heme with a high affinity and forms stable Hx-heme complexes. The heme–Hx complexes are readily endocytosed by macrophages expressing CD91 macroglobu‐ lin receptor, (also known as low-density lipoprotein receptor-related protein-1). Although under physiological conditions CD91 plays a role in recycling iron in response to extravascular hemolysis in hematoma-affected tissue, the Hx-heme/CD91 system may facilitate removal of prooxidative heme by icroglia/macrophages.

Recent studies and ongoing research in this laboratory support this notion and suggest that Hx-deficient mice experience augmented ICH injury. More studies of the role of Hx in ICH are warranted.
