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

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

intravascular hemolysis.

28 Intracerebral Hemorrhage

from the onset of ICH.

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

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

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.

prooxidative heme by icroglia/macrophages.

are warranted.

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

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 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 edema and the secondary brain damage after ICH.

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

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 chemicals capable of causing death to many brain cells.

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

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 in an HO-1 knockout mouse model leads to vulnerability to oxidant stress and inflammation. In the present study, intravenous administration of nicaraven (a marked synergistic induction of HO-1 protein, for 2 days after subarachnoid hemorrhage) ameliorated delayed cerebral vasospasm in rat subarachnoid hemorrhage models. These results suggest that the enhanced HO-1 expression through a combination of pathological state and pharmacological agent could be an effective strategy to improve the prognosis of heme- and oxidative stress-induced diseases. However, contradictory results have been reported in other researches. Overexpres‐ sion of HO-1 may be cytotoxic when excessive free iron exceeds the antioxidant properties of heme derived biliverdin. Earlier studies on ICH models in pigs and rabbits demonstrated that tin-mesoporphyrin, an isoform-nonselective HO inhibitor, attenuated edema formation and neuron loss. HO-1 and peroxidized lipid content were significantly higher in CSF from SAH patients with vasospasm, compared with nonvasospasm SAH CSF and correlated with occurrence of vasospasm. It is suggested that bilirubin and HO-1 was induced after hemor‐ rhagic stroke, reflecting the intensity of oxidative stress. One potential explanation for such discrepancy is that HO-1 deficiency could reduce the excessive liberation of free iron from erythrocytes in hematoma (feature that is unique to ICH) and consequently limit the ironmediated oxidative stress.After some controversy over the beneficial or toxic roles of HO-1, a better understanding of the pivotal HO-1 has evolved. We recently proposed a novel role of HO-1 in ICH pathogenesis. We concluded that upregulation of HO-1 after ICH may be a double-edged sword. The early mild upregulation possibly fit with the events and its overex‐ pression in the late stage may result in its dysfunction and be toxic. It should be prudent to intervene ICH with the inhibitor or activator of HO-1 and think over its potential dual effects.

month later). Lysis of RBCs and iron overload contribute to delayed edema formation after ICH. Iron accumulation in brain tissue is toxic and may result in brain damage after ICH. A recent clinical study showed that high serum levels of ferritin, a soluble protein for iron storage, are associated with poor outcome in ICH patients, suggesting that iron is involved in brain damage by ICH. Consistent with this idea, several studies on animal models of ICH showed that the iron chelator deferoxamine attenuated tissue damage and neurological deficits. For instance, intraperitoneal administration of deferoxamine starting from 2 or 6 h after autologous blood injection lessened brain edema, oxidative stress, and motor function deficits in rats. Another study on aged rats showed that deferoxamine was effective in reducing brain edema and motor dysfunction even when administration of the drug was delayed and 48 h, respec‐ tively, after autologous blood injection. However, the therapeutic efficacy of deferoxamine might depend on the type of ICH model. Reports mentioned above demonstrating beneficial effects of deferoxamine are based on the model made by autologous blood injection, whereas deferoxamine did not improve the outcome of the collagenase- induced ICH model in rat. On the other hand, a study on clioquinol, another kind of ferrous iron chelator, has demonstrated that oral administration of the drug, starting 6 h after induction of hemorrhage near the internal capsule by collagenase, alleviated motor dysfunction of rats . It recently has been demonstrated that estrogen reduces ferrous iron toxicity in vivo and in vitro, indicating that gender difference in susceptibility to ICH may, in part, be associated with differences in handling ferrous iron toxicity. Iron (II), by reacting with H2O2 generates hydroxyl radicals and clioquinol, by forming stable complexes with ferrous iron prevents its engagement in oxidative reactions. So , all these data suggest that iron particluarly ferrous iron (II) plays an important part in

The Pathogenesis of Edema and Secondary Insults after ICH

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31

Iron has the potential to mediate a number of deleterious reactions both in vitro and in vivo. Iron accumulation in tissues, particularly if the labile iron pool is increased, is associated with tissue damage.Iron overload in the brain can cause free-radical formation and oxidative damage such as lipid peroxidation after ICH. Brain cells, including neurons, astrocytes, and microglia, show a decreased ability to respond to oxidative stress, particularly with respect to their levels of glutathione and lutathione peroxidase, such that alteration in their iron status

Among the aquaporins(AQP) family, a major water-channel in the central nervous sys‐ tem(CNS )is AQP4, which is a key molecule for maintaining water balance, and its dysfunction or structural damage may cause brain edema.Aquaporin 4, a major water channel protein that is expressed in the brain, plays a key role in the maintenance of brain water homeostasis. It has been proposed that AQP4 may play an important role in the formation of cerebral edema. Because of restricted space within the cranium, salt and water fux in the CNS must be strictly regulated to maintain neuronal functions of the brain. In the CNS, most of the AQP4 is expressed in perimicrovessel astrocyte foot processes, and alterations in AQP4 expression are associated with perturbations of brain water homeostasis. The pattern of AQP4 expression was correlated with blood-brain barrier permeability, which was assessed using contrast enhanced Computed Tomography scanning. Our results showed that AQP4 was mainly located around blood vessels. The current study provided more direct evidence that AQP4 in perivascular

brain injury after ICH.

may predispose them to iron-induced oxidative stress.

**Figure 1.** Cerebral slice with autologus blood HO-1 positive staining in ICH rat in ICH rat

#### **4.2. Iron deposits and AQP4 [1-2, 5-6]**

As indicated, toxicity of free iron originated from extravascular hemolysis, and HO-mediated catabolism is well-documented. Iron derived from heme degradation may also play a key role in ICH pathogenesis, presumably via acceleration of oxidative stress. After autologous blood injection in rats, induction of HO-1 is followed by a gradual increase in tissue levels of nonheme iron. Iron concentrations in the brain can reach very high levels following RBC lysis, possibly contributing to acute brain edema formation (1st week) and delayed brain atrophy (1 month later). Lysis of RBCs and iron overload contribute to delayed edema formation after ICH. Iron accumulation in brain tissue is toxic and may result in brain damage after ICH. A recent clinical study showed that high serum levels of ferritin, a soluble protein for iron storage, are associated with poor outcome in ICH patients, suggesting that iron is involved in brain damage by ICH. Consistent with this idea, several studies on animal models of ICH showed that the iron chelator deferoxamine attenuated tissue damage and neurological deficits. For instance, intraperitoneal administration of deferoxamine starting from 2 or 6 h after autologous blood injection lessened brain edema, oxidative stress, and motor function deficits in rats. Another study on aged rats showed that deferoxamine was effective in reducing brain edema and motor dysfunction even when administration of the drug was delayed and 48 h, respec‐ tively, after autologous blood injection. However, the therapeutic efficacy of deferoxamine might depend on the type of ICH model. Reports mentioned above demonstrating beneficial effects of deferoxamine are based on the model made by autologous blood injection, whereas deferoxamine did not improve the outcome of the collagenase- induced ICH model in rat. On the other hand, a study on clioquinol, another kind of ferrous iron chelator, has demonstrated that oral administration of the drug, starting 6 h after induction of hemorrhage near the internal capsule by collagenase, alleviated motor dysfunction of rats . It recently has been demonstrated that estrogen reduces ferrous iron toxicity in vivo and in vitro, indicating that gender difference in susceptibility to ICH may, in part, be associated with differences in handling ferrous iron toxicity. Iron (II), by reacting with H2O2 generates hydroxyl radicals and clioquinol, by forming stable complexes with ferrous iron prevents its engagement in oxidative reactions. So , all these data suggest that iron particluarly ferrous iron (II) plays an important part in brain injury after ICH.

in an HO-1 knockout mouse model leads to vulnerability to oxidant stress and inflammation. In the present study, intravenous administration of nicaraven (a marked synergistic induction of HO-1 protein, for 2 days after subarachnoid hemorrhage) ameliorated delayed cerebral vasospasm in rat subarachnoid hemorrhage models. These results suggest that the enhanced HO-1 expression through a combination of pathological state and pharmacological agent could be an effective strategy to improve the prognosis of heme- and oxidative stress-induced diseases. However, contradictory results have been reported in other researches. Overexpres‐ sion of HO-1 may be cytotoxic when excessive free iron exceeds the antioxidant properties of heme derived biliverdin. Earlier studies on ICH models in pigs and rabbits demonstrated that tin-mesoporphyrin, an isoform-nonselective HO inhibitor, attenuated edema formation and neuron loss. HO-1 and peroxidized lipid content were significantly higher in CSF from SAH patients with vasospasm, compared with nonvasospasm SAH CSF and correlated with occurrence of vasospasm. It is suggested that bilirubin and HO-1 was induced after hemor‐ rhagic stroke, reflecting the intensity of oxidative stress. One potential explanation for such discrepancy is that HO-1 deficiency could reduce the excessive liberation of free iron from erythrocytes in hematoma (feature that is unique to ICH) and consequently limit the ironmediated oxidative stress.After some controversy over the beneficial or toxic roles of HO-1, a better understanding of the pivotal HO-1 has evolved. We recently proposed a novel role of HO-1 in ICH pathogenesis. We concluded that upregulation of HO-1 after ICH may be a double-edged sword. The early mild upregulation possibly fit with the events and its overex‐ pression in the late stage may result in its dysfunction and be toxic. It should be prudent to intervene ICH with the inhibitor or activator of HO-1 and think over its potential dual effects.

**Figure 1.** Cerebral slice with autologus blood HO-1 positive staining in ICH rat in ICH rat

As indicated, toxicity of free iron originated from extravascular hemolysis, and HO-mediated catabolism is well-documented. Iron derived from heme degradation may also play a key role in ICH pathogenesis, presumably via acceleration of oxidative stress. After autologous blood injection in rats, induction of HO-1 is followed by a gradual increase in tissue levels of nonheme iron. Iron concentrations in the brain can reach very high levels following RBC lysis, possibly contributing to acute brain edema formation (1st week) and delayed brain atrophy (1

**4.2. Iron deposits and AQP4 [1-2, 5-6]**

30 Intracerebral Hemorrhage

Iron has the potential to mediate a number of deleterious reactions both in vitro and in vivo. Iron accumulation in tissues, particularly if the labile iron pool is increased, is associated with tissue damage.Iron overload in the brain can cause free-radical formation and oxidative damage such as lipid peroxidation after ICH. Brain cells, including neurons, astrocytes, and microglia, show a decreased ability to respond to oxidative stress, particularly with respect to their levels of glutathione and lutathione peroxidase, such that alteration in their iron status may predispose them to iron-induced oxidative stress.

Among the aquaporins(AQP) family, a major water-channel in the central nervous sys‐ tem(CNS )is AQP4, which is a key molecule for maintaining water balance, and its dysfunction or structural damage may cause brain edema.Aquaporin 4, a major water channel protein that is expressed in the brain, plays a key role in the maintenance of brain water homeostasis. It has been proposed that AQP4 may play an important role in the formation of cerebral edema. Because of restricted space within the cranium, salt and water fux in the CNS must be strictly regulated to maintain neuronal functions of the brain. In the CNS, most of the AQP4 is expressed in perimicrovessel astrocyte foot processes, and alterations in AQP4 expression are associated with perturbations of brain water homeostasis. The pattern of AQP4 expression was correlated with blood-brain barrier permeability, which was assessed using contrast enhanced Computed Tomography scanning. Our results showed that AQP4 was mainly located around blood vessels. The current study provided more direct evidence that AQP4 in perivascular astroglial end feet plays a key role in exchange of water between brain, blood, and cerebro‐ spinal fuid. Upregulation of AQP4 induced by iron overload may cause an increased perme‐ ability to water in astrocytic membranes. The faint positive immunoreactivity of AQP4 possibly prevent the astrocytes from swelling. So, AQP4 in the brain may be viewed as a final common pathways of cerebral edema.

cord. Microglia are constantly scavenging the CNS for plaques, damaged neurons and infectious agents. The brain and spinal cord are considered "immune privileged" organs in that they are separated from the rest of the body by a series of endothelial cells known as the blood– brain barrier, which prevents most infections from reaching the vulnerable nervous tissue. In the case where infectious agents are directly introduced to the brain or cross the blood–brain barrier, microglial cells must react quickly to decrease inflammation and destroy the infectious agents before they damage the sensitive neural tissue. Due to the unavailability of antibodies from the rest of the body (few antibodies are small enough to cross the blood brain barrier), microglia must be able to recognize foreign bodies, swallow them, and act as antigen-present‐ ing cells activating T-cells. Since this process must be done quickly to prevent potentially fatal damage, microglia are extremely sensitive to even small pathological changes in the CNS.

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Microglia can be activated by a variety of factors including: glutamate receptor agonists, proinflammatory cytokines, cell necrosis factors, lipopolysaccharide, and changes in extracellular potassium (indicative of ruptured cells). Once activated the cells undergo several key mor‐ phological changes including the thickening and retraction of branches, uptake of major histocompatibility complex (MHC) class I/II proteins, expression of immunomolecules, secretion of cytotoxic factors, secretion of recruitment molecules, and secretion of proinflammatory signaling molecules (resulting in a pro-inflammation signal cascade). Activated non-phagocytic microglia generally appear as "bushy, " "rods, " or small ameboids depending on how far along the ramified to full phagocytic transformation continuum they are. In addition, the microglia also undergo rapid proliferation in order to increase their numbers. From a strictly morphological perspective, the variation in microglial form along the contin‐ uum is associated with changing morphological complexity and can be quantitated using the methods of fractal analysis, which have proven sensitive to even subtle, visually undetectable

changes associated with different morphologies in different pathological states.

Microglial cells are activated within minutes after the onset of ICH. The activated microglia release proinflammatory cytokines and chemotactic factors, which help to recruit hemotoge‐ neous inflammatory cells to the ICH injury sites. Activated microglial cells undergo morpho‐ logical and functional changes that include enlargement and thickening of processes, upregulation of proinflammatory proteins, and behavioral changes, including proliferation, migration and phagocytosis. Timely clearance of the extravasated RBCs by activated micro‐ glia/macrophages can provide protection from local damage resulting from RBC lysis. The primary neuroprotective role of activated microglia is to clear the hematoma and damaged cell debris through phagocytosis, providing a nurturing environment for tissue recovery. This is characterized first by the transient (18 hours–4 days) infiltration of neutrophils and then a long-term (1 day–months) presence of hematogenous macrophages. However, accumulating evidence has shown that microglial activation contributes to ICH-induced secondary brain injury by releasing a variety of cytokines, chemokines, free radicals, nitric oxide and other potentially toxic chemicals. In addition, several studies have shown that inhibition of micro‐ glial activation reduces brain damages in animal models of ICH. Microglial inhibitors, such as minocycline and microglia/macrophage inhibitory factors (tuftsin fragment 1–3), reduce ICHinduced brain injury and improve neurological function in rodents. Clearly, microglial activation mediates ICH-mediated brain injury. Successful removal of injured cells can reduce

**Figure 2.** Iron staining after ICH (Perl's staining) AQP4 positive staining in ICH rat
