**4. ICH animal models**

compromise and restore BBB integrity [88, 89]. This discrepancy could be explained by different injury models and different timing after injury. More work is needed to answer the

BM is a 3-dimensional network composed of extracellular matrix (ECM) proteins, including collagens, laminins, heparin sulfate proteoglycans, and nidogens [47, 90]. The formation of this network involves polymerization and cross-link of these ECM proteins [90, 91]. At the BBB, BMECs generate a vascular BM and astrocytes generate a parenchymal BM [92, 93]. The vascular and parenchymal BM is usually indistinguishable at capillaries [1]. However, at the post-capillary venules, the two BMs are separated by perivascular space where cerebrospinal fluid drains, and where antigen-presenting cells can be found [1]. Both BM layers have the same composition except that in the vascular BM laminin-α4 and-α5 are predominantly present [93], whereas in the parenchymal BM laminins-α1 and-α2 are the main components

Accumulating evidence suggests that loss of BM results in disruption of BBB, probably due to the loss of a physical barrier at the BMEC-astrocyte interface and/or lack of signaling from ECM molecules [95-99]. Individual ECM proteins, including laminin, collagen type IV, and fibronectin, have been shown to increase the TEER of BMECs *in vitro* [100]. Using laminin conditional knockout mice, we have shown that astrocytic laminin maintains BBB integrity by preventing pericyte differentiation from the resting stage to the contractile stage [101]. In addition, laminin α5 and dystroglycan, a major receptor for ECM proteins, have been found to negatively correlate with the infiltration of leukocytes in the brain [93]. These data suggest that BM plays a crucial role in BBB regulation. Future studies are expected to focus on the roles of individual ECM proteins in BBB integrity. Understanding how these ECM proteins affect individual BBB components and BBB integrity would significantly enhance our knowledge on

BBB and potentially pave the way for the treatment of many neurological disorders.

When ICH occurs, blood leaks into the brain parenchyma, leading to the formation of hema‐ toma, which quickly increases intracranial pressure. The accumulated blood and high intracranial pressure cause immediate primary damage to the brain. This initial injury is followed by secondary damage mainly resulting from inflammatory responses [102, 103]. The exposure of brain parenchyma to blood proteins (e.g., proteases and hemoglobin) and cells (red blood cells and leukocytes) results in activation of microglia, and the secretion of proinflammatory cytokines/chemokines [104, 105], including TNF-α, IL-1β, and MCP1/CCL2. These inflammatory mediators, by forming a concentration gradient, activate and attract more microglia and other inflammatory cells to the injury site [106]. These cells then accumulate around the hematoma, forming a barrier to prevent the spread of injury to other sites. The released pro-inflammatory cytokines/chemokines and possibly activated microglia also act on BMECs, pericytes and astrocytes, leading to compromise of BBB integrity. Through the

question how microglia regulate BBB integrity.

**f.** Basement Membrane (BM)

94 Intracerebral Hemorrhage

[92-94].

**3. Pathophysiology**

To study ICH and eventually cure this disease, several ICH animal models have been devel‐ oped, including collagenase ICH model, whole blood ICH model, and the spontaneous ICH model. Although these models have been widely used in ICH research, none of them fully replicates the pathology of ICH in human patients. Here we briefly discuss the advantages and disadvantages of these models.

#### **a.** Collagenase ICH Model

This model utilizes the enzymatic activity of collagenase, a bacterial enzyme. After injection into the brain, collagenase induces rupture of blood vessels by degrading collagen IV, a component of the blood vessel wall [103-105]. The rupture of blood vessels then induces the formation of hematoma and other pathological alterations. There are many advantages of this model. First, ICH induced by collagenase injection is very reliable and reproducible. The size and location of hematoma reported by different laboratories across the world are comparable [112-115]. Second, the location of hematoma can be controlled depending on the site of injection. Third, this model is very simple and fast. ICH can be induced within hours after collagenase injection. Due to these advantages, collagenase ICH model has become one of the most popular animal models for ICH research. This model, however, also has a few disad‐ vantages. One of the most significant drawbacks is that it introduces collagenase, a bacterial enzyme, into the mammalian brain. This enzyme degrades ECM proteins in the brain, affects BBB integrity, and modifies inflammatory or immune responses, all of which may affect ICH progress [105, 116, 117]. Another disadvantage of this model is that it does not replicate the vascular challenges usually seen before the onset of ICH in patients, such as hypertension and atherosclerosis. Mice lacking these vascular injuries may have different disease progress and/ or recovery patterns, which makes it difficult to interpret data generated using this ICH model.

**b.** Whole Blood ICH Model

The whole blood model involves injection of blood from the same animal or a donor into the brain. The injected blood induces secondary pathological changes observed in human patients. Unlike the collagenase ICH model, this model does not introduce exogenous enzymes. The application of this ICH model, however, is circumvented by its three major disadvantages. First, the whole blood ICH model lacks pathological changes in blood vessels. The vascular challenges and rupture of vasculature cannot be replicated in this model. Second, this model is less reproducible than collagenase ICH model. The size and location of hematoma vary depending on different laboratories. Third, the shape of hematoma is different from that found in human patients. Hematoma formed in whole blood ICH model is usually umbrella-shaped and narrower slit-like [118]. This unique shape is probably caused by high pressure-induced rapid distribution of blood along white matter tracts and/or corpus callosum after injection. A way to get around this problem is used in bigger animals, like pigs, where a space/balloon forming initial injection is followed by the injection of the homologous whole blood.

115], inhibition of microglial activation has been proposed as a therapeutic strategy for ICH. It has been shown that pre-or post-treatment with the tri-peptide microglia/macrophage inhibitory factor (MIF, Thr-Lys-Pro) significantly inhibited microglial activation, reduced injury size and improved neurological function [104, 105]. Consistently with this report, inhibiting microglial activation with neuroprotectant minocycline in both collagenase and whole blood ICH models protected BBB integrity, decreased brain edema, and improved functional recovery, although neuronal death remained changed [126-130]. These data support that inhibition of microglial activation is beneficial. However, there is also evidence suggesting that long-term inhibition of microglial activation is detrimental [104, 115]. Given that activated microglia also contribute to the clearance of cell debris and recovery at late stage, inhibition of microglial activation should be limited to the early stage. The question then becomes how to define early and late stages after ICH? Definition of these stages would significantly improve

Recovery from ICH – Potential Targets http://dx.doi.org/10.5772/58477 97

Leukocytes infiltrate into the brain through the compromised BBB and modulate the progress and/or recovery of ICH [102, 112]. Among all the subtypes of leukocytes, neutrophils are the earliest ones to infiltrate into the brain after ICH. In both collagenase and whole blood ICH models in rodents, neutrophil infiltration starts at approximately 4 hours and peaks at 3 days after the onset of ICH [102, 115, 124, 131, 132]. These cells promote cell death and brain damage by producing ROS and pro-inflammatory mediators [107, 108], and usually die within 2 days in the brain. Mice deficient for CD18, a subunit of β2 integrin indispensable for leukocyte infiltration, demonstrated reduced brain edema and mortality as well as decreased leukocyte number in the brain after collagenase injection [133]. In human postmortem brains, leukocyte infiltration was also observed within hours after ICH [134, 135]. Furthermore, leukocyte counts in blood have been found to positively correlate with injury size in ICH patients [136]. Therefore, high leukocyte counts together with other factors have been used to predict early clinical outcome in ICH patients [137, 138]. Currently, no anti-leukocyte infiltration strategies have been investigated in ICH models. Obtaining such data may facilitate the research and

During ICH, activated microglia and infiltrated leukocytes produce high levels of inflamma‐ tory cytokines/chemokines, which mediate the secondary damage to the brain. In both rodents and humans, pro-inflammatory cytokines, including TNF-α and IL-1β, are transiently upregulated in the peri-hematomal region [106, 139]. In addition, chemokines and chemokine receptors that mediate leukocyte extravasation, including CCL2-4, IL-8, CXCL5, and CCR1-2, are also increased/activated [139, 140]. These data suggest that targeting cytokine/chemokine signaling may be a therapeutic strategy for ICH. In collagenase ICH model, we have found that mice deficient for CCL2 or its receptor CCR2 have a mild but delayed disease progression [115]. In CCL2-/-or CCR2-/-mice, hematoma was smaller at day 1 post injury (dpi 1) but larger at subsequent times (dpi 7 and 14 [115]), indicating a delayed recovery. Consistent with the crucial role of CCL2-CCR2 system in microglial activation/migration, limited numbers of

development of novel reagents targeting leukocyte infiltration.

the outcome of ICH treatments.

**b.** Leukocyte Infiltration

**c.** Cytokines/Chemokines

#### **c.** Spontaneous ICH Model

To better replicate the pathological changes observed in human patients, a spontaneous ICH model has been developed in rodents [119]. This new model induces ICH through acute hypertension, the most common etiology of hemorrhage in humans. In this model, animals are administered with NG-nitro-L-arginine methyl ester (L-NAME) and angiotensin II to induce hypertension. The injection of angiotensin II causes surges of blood pressure, which eventually lead to rupture of blood vessels and thus ICH. This spontaneous ICH model replicates most pathological alterations observed in human patients. However, the time it takes to induce ICH is relatively long (2-4 weeks), the location of the ICH varies, and the reprodu‐ cibility still needs further investigation.

#### **5. Targets for ICH treatments**

ICH is a devastating clinical event. Sadly, no effective treatments are available at present. Current therapy is mainly supportive care [120, 121]. Due to the pivotal role of inflammatory responses in ICH development, anti-inflammatory strategies have been explored by many laboratories. Here we review a few anti-inflammatory targets with therapeutic potential in ICH: microglial activation, leukocyte infiltration, cytokines/chemokines, protease activation, and reactive oxygen species (ROS) production. In addition, stem cell therapy is also discussed briefly.

**a.** Microglial Activation

Microglia are one of the first cell types that respond to ICH. In collagenase ICH model, microglial activation starts at 1 hour [102, 122], peaks at 3-7 days [104, 105, 115, 123], and returns to a resting state again by 3-4 weeks after the onset of ICH [124, 125]. A similar time course of microglial activation is observed in whole blood ICH model [122, 124, 125]. Since activated microglia contribute to the amplification of inflammatory responses and cell death by secreting chemotactic cytokines and cytotoxic mediators, including proteases and ROS [102, 103, 112, 115], inhibition of microglial activation has been proposed as a therapeutic strategy for ICH. It has been shown that pre-or post-treatment with the tri-peptide microglia/macrophage inhibitory factor (MIF, Thr-Lys-Pro) significantly inhibited microglial activation, reduced injury size and improved neurological function [104, 105]. Consistently with this report, inhibiting microglial activation with neuroprotectant minocycline in both collagenase and whole blood ICH models protected BBB integrity, decreased brain edema, and improved functional recovery, although neuronal death remained changed [126-130]. These data support that inhibition of microglial activation is beneficial. However, there is also evidence suggesting that long-term inhibition of microglial activation is detrimental [104, 115]. Given that activated microglia also contribute to the clearance of cell debris and recovery at late stage, inhibition of microglial activation should be limited to the early stage. The question then becomes how to define early and late stages after ICH? Definition of these stages would significantly improve the outcome of ICH treatments.

#### **b.** Leukocyte Infiltration

The whole blood model involves injection of blood from the same animal or a donor into the brain. The injected blood induces secondary pathological changes observed in human patients. Unlike the collagenase ICH model, this model does not introduce exogenous enzymes. The application of this ICH model, however, is circumvented by its three major disadvantages. First, the whole blood ICH model lacks pathological changes in blood vessels. The vascular challenges and rupture of vasculature cannot be replicated in this model. Second, this model is less reproducible than collagenase ICH model. The size and location of hematoma vary depending on different laboratories. Third, the shape of hematoma is different from that found in human patients. Hematoma formed in whole blood ICH model is usually umbrella-shaped and narrower slit-like [118]. This unique shape is probably caused by high pressure-induced rapid distribution of blood along white matter tracts and/or corpus callosum after injection. A way to get around this problem is used in bigger animals, like pigs, where a space/balloon

forming initial injection is followed by the injection of the homologous whole blood.

To better replicate the pathological changes observed in human patients, a spontaneous ICH model has been developed in rodents [119]. This new model induces ICH through acute hypertension, the most common etiology of hemorrhage in humans. In this model, animals are administered with NG-nitro-L-arginine methyl ester (L-NAME) and angiotensin II to induce hypertension. The injection of angiotensin II causes surges of blood pressure, which eventually lead to rupture of blood vessels and thus ICH. This spontaneous ICH model replicates most pathological alterations observed in human patients. However, the time it takes to induce ICH is relatively long (2-4 weeks), the location of the ICH varies, and the reprodu‐

ICH is a devastating clinical event. Sadly, no effective treatments are available at present. Current therapy is mainly supportive care [120, 121]. Due to the pivotal role of inflammatory responses in ICH development, anti-inflammatory strategies have been explored by many laboratories. Here we review a few anti-inflammatory targets with therapeutic potential in ICH: microglial activation, leukocyte infiltration, cytokines/chemokines, protease activation, and reactive oxygen species (ROS) production. In addition, stem cell therapy is also discussed

Microglia are one of the first cell types that respond to ICH. In collagenase ICH model, microglial activation starts at 1 hour [102, 122], peaks at 3-7 days [104, 105, 115, 123], and returns to a resting state again by 3-4 weeks after the onset of ICH [124, 125]. A similar time course of microglial activation is observed in whole blood ICH model [122, 124, 125]. Since activated microglia contribute to the amplification of inflammatory responses and cell death by secreting chemotactic cytokines and cytotoxic mediators, including proteases and ROS [102, 103, 112,

**c.** Spontaneous ICH Model

96 Intracerebral Hemorrhage

cibility still needs further investigation.

**5. Targets for ICH treatments**

briefly.

**a.** Microglial Activation

Leukocytes infiltrate into the brain through the compromised BBB and modulate the progress and/or recovery of ICH [102, 112]. Among all the subtypes of leukocytes, neutrophils are the earliest ones to infiltrate into the brain after ICH. In both collagenase and whole blood ICH models in rodents, neutrophil infiltration starts at approximately 4 hours and peaks at 3 days after the onset of ICH [102, 115, 124, 131, 132]. These cells promote cell death and brain damage by producing ROS and pro-inflammatory mediators [107, 108], and usually die within 2 days in the brain. Mice deficient for CD18, a subunit of β2 integrin indispensable for leukocyte infiltration, demonstrated reduced brain edema and mortality as well as decreased leukocyte number in the brain after collagenase injection [133]. In human postmortem brains, leukocyte infiltration was also observed within hours after ICH [134, 135]. Furthermore, leukocyte counts in blood have been found to positively correlate with injury size in ICH patients [136]. Therefore, high leukocyte counts together with other factors have been used to predict early clinical outcome in ICH patients [137, 138]. Currently, no anti-leukocyte infiltration strategies have been investigated in ICH models. Obtaining such data may facilitate the research and development of novel reagents targeting leukocyte infiltration.

#### **c.** Cytokines/Chemokines

During ICH, activated microglia and infiltrated leukocytes produce high levels of inflamma‐ tory cytokines/chemokines, which mediate the secondary damage to the brain. In both rodents and humans, pro-inflammatory cytokines, including TNF-α and IL-1β, are transiently upregulated in the peri-hematomal region [106, 139]. In addition, chemokines and chemokine receptors that mediate leukocyte extravasation, including CCL2-4, IL-8, CXCL5, and CCR1-2, are also increased/activated [139, 140]. These data suggest that targeting cytokine/chemokine signaling may be a therapeutic strategy for ICH. In collagenase ICH model, we have found that mice deficient for CCL2 or its receptor CCR2 have a mild but delayed disease progression [115]. In CCL2-/-or CCR2-/-mice, hematoma was smaller at day 1 post injury (dpi 1) but larger at subsequent times (dpi 7 and 14 [115]), indicating a delayed recovery. Consistent with the crucial role of CCL2-CCR2 system in microglial activation/migration, limited numbers of microglia were observed at dpi 1 in both knockout mice [115]. At dpi 3 and 7, however, the number of microglia in the knockout mice far exceeded those in control animals [115], suggesting that CCL2-CCR2 independent alternative signaling recruited microglia in the knockout mice. The infiltration of neutrophils was also ablated in both knockout mice at dpi 1 and 3, echoed by the smaller hematoma size early after injury [115]. In addition, at dpi 7 the expression of inducible nitric oxide synthase (iNOS) decreased in controls compared to earlier time-points, but remained high in the mutant mice, indicating that lack of CCL2-CCR2 signaling produces more ROS. Moreover, brain edema, neuronal loss and neurological function followed similar trends over time as that of hematoma size [115]. Altogether, these data suggest that inhibiting CCL2-CCR2 signaling early after ICH is neuroprotective, whereas long-term inhibition delays the recovery. Future work should focus on developing the best CCL2-CCR2 inhibition regimen for ICH patients.

Accumulated iron has been shown to induce oxidative stress by formation of free radicals, mediate secondary inflammatory injury, and contribute to brain atrophy and neurological deficits after ICH [157, 158]. In human patients with spontaneous ICH, blood ferritin level associates with brain edema in peri-hematomal region [159]. In addition, iron level in the hematoma also correlates with brain edema in peri-hematomal area [160]. These data suggest that iron deposition contributes to brain damage, and that removing the deposited iron may be an appropriate therapeutic approach. Consistent with this hypothesis, 2, 2'-dipyridyl, a lipid-soluble iron chelator, has been shown to be beneficial in both the collagenase and whole blood ICH models in mice [161]. Another iron chelator deferoxamine has shown neuropro‐ tective effects in the whole blood ICH model in rats and piglets [162-165]. In collagenase ICH model, however, deferoxamine failed to show any beneficial effects [166], suggesting the effect of deferoxamine depends on ICH animal models. None-the-less high doses of deferoxamine

Recovery from ICH – Potential Targets http://dx.doi.org/10.5772/58477 99

are currently examined in clinical trials (starting in 2012) for the treatment of ICH.

potential.

**6. Summary**

**f.** Stem Cell Therapy

effects of stem cell administration must be examined.

An alternative way to treat iron-induced oxidative stress is to target antioxidant enzymes. To remove extra ROS, antioxidant enzymes, including glutathione S transferases, glutathione peroxidase, and glutamate-cysteine ligase, are up-regulated. The key transcription factor that controls the expression of these antioxidant enzymes is Nrf2 [167]. Nrf2 is expressed in neuronal and glial cells in the brain. Activation of Nrf2 has been shown to be neuroprotective both *in vitro* and *in vivo* [168, 169]. Additionally, mice deficient for Nrf2 showed more severe neurological deficits compared to wild-type mice in both collagenase and whole blood ICH models [170, 171]. Paralleled with neurological deficits, enhanced ROS production and leukocyte infiltration were observed in Nrf2-/-mice [170, 171]. More importantly, sulforaphane, an Nrf2 inducer, has been reported to improve neurological deficits in mice when administered 30 minutes after ICH [170]. Together, these data suggest that Nrf2 is a target with therapeutic

ICH induces neuronal death and loss of neurological function. Multipotent stem cells with the ability to differentiate into neurons are a potential therapy for ICH. It has been reported that human neural stem cells are able to differentiate into neurons and astrocytes, and thus improve neurological function after intravenous injection in collagenase ICH model [172]. Stem cell therapy is relatively new and more work is needed before it can be used in ICH patients. For example, the route, dose and timing of stem cell injection need to be optimized; the differen‐ tiation, proliferation and integration of stem cells *in vivo* should be investigated; and the side

Accumulating evidence suggests that the secondary inflammatory responses play a critical role in the development of ICH, indicating that the molecular mechanism of inflammation is an ideal target for the therapy of ICH. As discussed, many pathways, including microglia activation, leukocyte infiltration, cytokine/chemokine secretion, protease activation, and ROS

#### **d.** Protease Activation

ICH activates many proteases, including matrix metalloproteinases (MMPs). MMPs are a group of zinc-dependent proteases actively involved in extracellular remodeling and neuro‐ inflammation. Under physiological conditions, low levels of inactive MMPs are found in the brain. These MMPs, however, are dramatically up-regulated and activated when ICH occurs [112, 141]. We and others have demonstrated that collagenase quickly activates and upregulates the expression of MMP-2,-3,-9, and-12 in rodents [112, 142]. Activation of MMP-9 has also been described in other ICH models [143-145]. In human ICH patients, blood MMP-9 level has been reported to correlate with BBB integrity, hematoma size, edema of the penumbra area, and neurological function [138, 146, 147], whereas blood MMP-3 levels have been found to associate with mortality [148]. Additionally, higher level of MMP-9 was detected in the perihematomal region in postmortem human brains [113, 149]. These data suggest that modulation of MMP activity may have therapeutic effect in ICH. Consistent with this hypothesis, mice lacking MMP-3,-9, or-12 are partially protected from ICH [141, 144, 150]. In addition, the therapeutic effect of MMP inhibitors has also been investigated. GM6001, a broad-spectrum MMP inhibitor, has been found to be neuroprotective in both collagenase and whole blood ICH models in mice [132, 151]. Similar results have been noted for BB-1101, another broadspectrum MMP inhibitor [152]. However, both neuroprotective and detrimental roles have been reported for MMP inhibitor BB-94, depending on the animal models used [153-155]. Besides its inhibitory effect on microglial activation, minocycline also functions as a MMP inhibitor [126]. There is evidence suggesting that minocycline reduces TNF-α level and brain edema without affecting neuronal loss [127, 156], when administered 6 hours after ICH. Together, these data suggest that MMPs, especially MMP-9, play a detrimental role in ICH, and that MMP inhibitors may be used, alone or in combination with other medicine, to treat ICH.

#### **e.** ROS Production

One of the main pathological changes of ICH is the accumulation of blood in the brain. The hemolysis of extravasated red blood cells leads to degradation of hemoglobin and deposition of iron in the brain [109]. In rats, a 3-fold increase of non-heme iron was found after ICH [109]. Accumulated iron has been shown to induce oxidative stress by formation of free radicals, mediate secondary inflammatory injury, and contribute to brain atrophy and neurological deficits after ICH [157, 158]. In human patients with spontaneous ICH, blood ferritin level associates with brain edema in peri-hematomal region [159]. In addition, iron level in the hematoma also correlates with brain edema in peri-hematomal area [160]. These data suggest that iron deposition contributes to brain damage, and that removing the deposited iron may be an appropriate therapeutic approach. Consistent with this hypothesis, 2, 2'-dipyridyl, a lipid-soluble iron chelator, has been shown to be beneficial in both the collagenase and whole blood ICH models in mice [161]. Another iron chelator deferoxamine has shown neuropro‐ tective effects in the whole blood ICH model in rats and piglets [162-165]. In collagenase ICH model, however, deferoxamine failed to show any beneficial effects [166], suggesting the effect of deferoxamine depends on ICH animal models. None-the-less high doses of deferoxamine are currently examined in clinical trials (starting in 2012) for the treatment of ICH.

An alternative way to treat iron-induced oxidative stress is to target antioxidant enzymes. To remove extra ROS, antioxidant enzymes, including glutathione S transferases, glutathione peroxidase, and glutamate-cysteine ligase, are up-regulated. The key transcription factor that controls the expression of these antioxidant enzymes is Nrf2 [167]. Nrf2 is expressed in neuronal and glial cells in the brain. Activation of Nrf2 has been shown to be neuroprotective both *in vitro* and *in vivo* [168, 169]. Additionally, mice deficient for Nrf2 showed more severe neurological deficits compared to wild-type mice in both collagenase and whole blood ICH models [170, 171]. Paralleled with neurological deficits, enhanced ROS production and leukocyte infiltration were observed in Nrf2-/-mice [170, 171]. More importantly, sulforaphane, an Nrf2 inducer, has been reported to improve neurological deficits in mice when administered 30 minutes after ICH [170]. Together, these data suggest that Nrf2 is a target with therapeutic potential.

**f.** Stem Cell Therapy

microglia were observed at dpi 1 in both knockout mice [115]. At dpi 3 and 7, however, the number of microglia in the knockout mice far exceeded those in control animals [115], suggesting that CCL2-CCR2 independent alternative signaling recruited microglia in the knockout mice. The infiltration of neutrophils was also ablated in both knockout mice at dpi 1 and 3, echoed by the smaller hematoma size early after injury [115]. In addition, at dpi 7 the expression of inducible nitric oxide synthase (iNOS) decreased in controls compared to earlier time-points, but remained high in the mutant mice, indicating that lack of CCL2-CCR2 signaling produces more ROS. Moreover, brain edema, neuronal loss and neurological function followed similar trends over time as that of hematoma size [115]. Altogether, these data suggest that inhibiting CCL2-CCR2 signaling early after ICH is neuroprotective, whereas long-term inhibition delays the recovery. Future work should focus on developing the best

ICH activates many proteases, including matrix metalloproteinases (MMPs). MMPs are a group of zinc-dependent proteases actively involved in extracellular remodeling and neuro‐ inflammation. Under physiological conditions, low levels of inactive MMPs are found in the brain. These MMPs, however, are dramatically up-regulated and activated when ICH occurs [112, 141]. We and others have demonstrated that collagenase quickly activates and upregulates the expression of MMP-2,-3,-9, and-12 in rodents [112, 142]. Activation of MMP-9 has also been described in other ICH models [143-145]. In human ICH patients, blood MMP-9 level has been reported to correlate with BBB integrity, hematoma size, edema of the penumbra area, and neurological function [138, 146, 147], whereas blood MMP-3 levels have been found to associate with mortality [148]. Additionally, higher level of MMP-9 was detected in the perihematomal region in postmortem human brains [113, 149]. These data suggest that modulation of MMP activity may have therapeutic effect in ICH. Consistent with this hypothesis, mice lacking MMP-3,-9, or-12 are partially protected from ICH [141, 144, 150]. In addition, the therapeutic effect of MMP inhibitors has also been investigated. GM6001, a broad-spectrum MMP inhibitor, has been found to be neuroprotective in both collagenase and whole blood ICH models in mice [132, 151]. Similar results have been noted for BB-1101, another broadspectrum MMP inhibitor [152]. However, both neuroprotective and detrimental roles have been reported for MMP inhibitor BB-94, depending on the animal models used [153-155]. Besides its inhibitory effect on microglial activation, minocycline also functions as a MMP inhibitor [126]. There is evidence suggesting that minocycline reduces TNF-α level and brain edema without affecting neuronal loss [127, 156], when administered 6 hours after ICH. Together, these data suggest that MMPs, especially MMP-9, play a detrimental role in ICH, and that MMP inhibitors may be used, alone or in combination with other medicine, to treat

One of the main pathological changes of ICH is the accumulation of blood in the brain. The hemolysis of extravasated red blood cells leads to degradation of hemoglobin and deposition of iron in the brain [109]. In rats, a 3-fold increase of non-heme iron was found after ICH [109].

CCL2-CCR2 inhibition regimen for ICH patients.

**d.** Protease Activation

98 Intracerebral Hemorrhage

ICH.

**e.** ROS Production

ICH induces neuronal death and loss of neurological function. Multipotent stem cells with the ability to differentiate into neurons are a potential therapy for ICH. It has been reported that human neural stem cells are able to differentiate into neurons and astrocytes, and thus improve neurological function after intravenous injection in collagenase ICH model [172]. Stem cell therapy is relatively new and more work is needed before it can be used in ICH patients. For example, the route, dose and timing of stem cell injection need to be optimized; the differen‐ tiation, proliferation and integration of stem cells *in vivo* should be investigated; and the side effects of stem cell administration must be examined.

### **6. Summary**

Accumulating evidence suggests that the secondary inflammatory responses play a critical role in the development of ICH, indicating that the molecular mechanism of inflammation is an ideal target for the therapy of ICH. As discussed, many pathways, including microglia activation, leukocyte infiltration, cytokine/chemokine secretion, protease activation, and ROS production, have been explored, and several compounds showed significant potential in the treatment of ICH. However, it should be noted that the animal models used in the studies are not perfect, which limits the interpretation of experimental data. Thus, other models and human samples should be used to confirm the results before they are used in patients.

[8] Benchenane K, Lopez-Atalaya JP, Fernandez-Monreal M, Touzani O, Vivien D. Equivocal roles of tissue-type plasminogen activator in stroke-induced injury. Trends

Recovery from ICH – Potential Targets http://dx.doi.org/10.5772/58477 101

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## **Author details**

Yao Yao1,2 and Stella E. Tsirka1\*

\*Address all correspondence to: stella@pharm.stonybrook.edu

1 Program in Molecular and Cellular Pharmacology, Department of Pharmacological Scien‐ ces, Stony Brook University, Stony Brook, NY, USA

2 Laboratory of Neurobiology and Genetics, The Rockefeller University, New York, NY, USA

## **References**


[8] Benchenane K, Lopez-Atalaya JP, Fernandez-Monreal M, Touzani O, Vivien D. Equivocal roles of tissue-type plasminogen activator in stroke-induced injury. Trends Neurosci 2004; 27:155-160.

production, have been explored, and several compounds showed significant potential in the treatment of ICH. However, it should be noted that the animal models used in the studies are not perfect, which limits the interpretation of experimental data. Thus, other models and human samples should be used to confirm the results before they are used in patients.

1 Program in Molecular and Cellular Pharmacology, Department of Pharmacological Scien‐

2 Laboratory of Neurobiology and Genetics, The Rockefeller University, New York, NY,

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