Collagenase-induced Intracerebral Hemorrhage Model Research Articles

P2X7 Receptor Suppression Preserves Blood-Brain Barrier through Inhibiting RhoA Activation after Experimental Intracerebral Hemorrhage in Rats.

Blockading P2X7 receptor(P2X7R) provides neuroprotection toward various neurological disorders, including stroke, traumatic brain injury, and subarachnoid hemorrhage. However, whether and how P2X7 receptor suppression protects blood-brain barrier(BBB) after intracerebral hemorrhage(ICH) remains unexplored. In present study, intrastriatal autologous-blood injection was used to mimic ICH in rats. Selective P2X7R inhibitor A438079, P2X7R agonist BzATP, and P2X7R siRNA were administrated to evaluate the effects of P2X7R suppression. Selective RhoA inhibitor C3 transferase was administered to clarify the involvement of RhoA. Post-assessments, including neurological deficits, Fluoro-Jade C staining, brain edema, Evans blue extravasation and fluorescence, western blot, RhoA activity assay and immunohistochemistry were performed. Then the key results were verified in collagenase induced ICH model. We found that endogenous P2X7R increased at 3 hrs after ICH with peak at 24 hrs, then returned to normal at 72 hrs after ICH. Enhanced immunoreactivity was observed on the neurovascular structure around hematoma at 24 hrs after ICH, along with perivascular astrocytes and endothelial cells. Both A438079 and P2X7R siRNA alleviated neurological deficits, brain edema, and BBB disruption after ICH, in association with RhoA activation and down-regulated endothelial junction proteins. However, BzATP abolished those effects. In addition, C3 transferase reduced brain injury and increased endothelial junction proteins’ expression after ICH. These data indicated P2X7R suppression could preserve BBB integrity after ICH through inhibiting RhoA activation.

Autophagy regulates intracerebral hemorrhage induced neural damage via apoptosis and NF-κB pathway

Autophagy can be a pro-survival or a pro-death mechanism depending on the context. The role of autophagy in intracerebral hemorrhage (ICH) remains elusive. In this study, in vivo and in vitro experiments have been carried out to investigate the role of autophagy after ICH. Collagenase-induced ICH model in mouse was made for in vivo experiments. Primary cortical neurons cultures were exposed to hemin to mimic ICH in vitro. 3-Methyladenine (3-MA) and rapamycin (RAP) were administrated both in vivo and in vitro. We first measured brain water content and cell death after ICH in model. Expression of LC3, p62/SQSTM1 (p62), Beclin1, Caspase3 and Bcl-2, which have been found related to autophagy and apoptosis, were assessed both in vivo and in vitro. Furthermore, NF-κB was detected to explore the potential mechanisms. We found brain edema in ICH model in mouse and the number of Propidium Iodide (PI)-positive cells both in vivo and in vitro were decreased by 3-MA pretreated. Simultaneously, both in vivo and in vitro, 3-MA significantly decreased the expression of LC3-II and Beclin-1, and maintained p62 at high level after ICH. Furthermore, pretreatment with 3-MA downregulated the level of cleaved caspase-3 but upregulated the Bcl-2 level. Conversely, RAP pretreatment reversed all these results above. These data indicated that autophagy activation may deprave ICH induced brain injury in ICH model and neuro-damage may be related to regulating of NF-κB pathway and thereby promote inflammation and apoptosis, thus might provide novel therapeutic interventions for ICH.

Brain recovery mediated by toll-like receptor 4 in rats after intracerebral hemorrhage

Activation of the immune system via toll-like receptor 4 (TLR4) is implicated in both negative and positive processes in the central nervous system, including inflammation and angiogenesis. Whether TLR4 also participates in brain recovery following intracerebral hemorrhage (ICH) has not been investigated. We used the rat model of collagenase-induced ICH to determine whether TLR4 acts as a key regulator of brain recovery in the late phase of injury. After ICH, TLR4 levels in the ipsilateral striatum were significantly higher in the ICH group than in the Sham group on days 1, 3, 7 and 14 after ICH induction. By 14 d, the ICH group showed significantly higher levels of vascular endothelial growth factor, brain-derived neurotrophic factor, and MMP-9 than the Sham group, as well as greater numbers of vessels and BrdU- and DCX-positive cells. All these ICH-induced increases were significantly smaller in the TAK-242 group. The TLR4 antagonist also inhibited the recovery of neurological function after ICH. A TLR4 antagonist reduced ICH-induced neurogenesis and angiogenesis in a rat. These findings suggest that TLR4 may promote brain repair in the late phase of ICH.

Anti-inflammatory activities of carbon monoxide-releasing molecules (CO-RMs) in the brain.

The transcription factor Nrf2 and its downstream target heme oxygenase-1 (HO-1) are ubiquitous protective systems against oxidative stress, inflammation and tissue injury. The products of HO-1 enzymatic activity, biliverdin and carbon monoxide (CO), possess interesting anti-oxidant and anti-inflammatory properties suggesting that exploitation of this pathway as a target for drug discovery may offer therapeutic avenues in a variety of disorders [1]. In this context we have developed carbon monoxide-releasing molecules (CO-RMs), a class of compounds that deliver CO in a controlled fashion and exert a variety of pharmacological effects [2]. Data will be presented showing that CO-RMs modulate neuroinflammation and neuroprotection in vitro and in vivo. In BV2 microglia cells challenged with thrombin and interferon gamma, CO-RMs reduced the production of inflammatory mediators both in normoxic and hypoxic conditions. Similarly, in a rat model of collagenase-induced intracerebral hemorrhage, CO-RMs modulated microglia activation and the production of TNF-α while partially protecting against brain damage [3]. The mechanism(s) by which CO liberated from CO-RMs exert protection remains elusive. However, our laboratory has recently produced findings in BV2 microglia pointing to mitochondria as a plausible target for the anti-inflammatory action of CO. Studies are also currently ongoing on the synthesis and characterization of novel hybrid molecules that potently activate Nrf2 and simultaneously release CO in order to maximize the anti-inflammatory effects of the HO-1 pathway [4].

HMGB1 may act via RAGE to promote angiogenesis in the later phase after intracerebral hemorrhage

Following intracerebral hemorrhage (ICH), high-mobility group box 1 protein (HMGB1) may promote vascular remodeling. Whether HMGB1 supports angiogenesis after ICH is unclear, as are the receptors and downstream signaling pathway(s) involved. We used the rat model of collagenase-induced ICH to determine whether HMGB1 acts via the receptor for advanced glycation end-products (RAGE) to upregulate vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells and key regulator of normal and abnormal angiogenesis in the late phase of injury. At 3d after ICH induction, rats were treated with saline, ethyl pyruvate (EP) or N-benzyl-4-chloro-N-cyclohexylbenzamide (FPS-ZM1). ICH induced the movement of HMGB1 from the nucleus into the cytoplasm. Levels of HMGB1 and RAGE in the ipsilateral striatum increased within a few days of induction and continued to rise for 7–14d afterward. By 14d after induction, levels of VEGF and vessel density were higher than in the Sham group. Administering EP 3days after ICH induction prevented much of the stroke-induced increases in vessel density and in expression of HMGB1, RAGE, and VEGF. Administering FPS-ZM1 after ICH blocked much of the stroke-induced increases in vessel density and VEGF expression. Our results suggest that after ICH, HMGB1 may upregulate VEGF in the ipsilateral striatum predominantly via RAGE. Hence, targeting the HMGB1/RAGE signaling pathway may help reduce inappropriate angiogenesis after ICH.

Necrostatin-1 Ameliorates Intracerebral Hemorrhage-Induced Brain Injury in Mice Through Inhibiting RIP1/RIP3 Pathway

Necroptosis is a recently discovered programmed necrosis, regulated by receptor interacting protein kinase 1 (RIP1) and RIP3 after death signal stimulation and could be specifically inhibited by necrostatin-1. The aim of this study was to investigate the role of RIP1 and RIP3 signal pathways in a mouse model of collagenase-induced intracerebral hemorrhage (ICH) and assess the effect of necrostatin-1 on brain injury after ICH. We found that RIP1 and RIP3 proteins were abundantly expressed and increased in mice brain after ICH. Necrostatin-1 pretreatment improved neurological function and attenuated brain edema in mice after ICH. Moreover, necrostatin-1 reduced RIP1-RIP3 interaction and propidium iodide (PI) positive cell death, and further inhibited microglia activation and pro-inflammatory mediator genes [tumor necrosis factor-a (TNF-α) and interleukin-1β (IL-1β)] expression after ICH. These findings indicate that RIP1/RIP3-mediated necroptosis is an important mechanism of cell death after ICH. Through inhibiting necroptosis, necrostatin-1 plays a protective role in reducing necrotic cell death after ICH. Necrostatin-1 is a promising therapeutic agent that protects cells from necroptosis and improves functional outcome.

PGE2 receptor agonist misoprostol protects brain against intracerebral hemorrhage in mice

Intracerebral hemorrhage (ICH) is a devastating form of stroke. Misoprostol, a synthetic prostaglandin E1 (PGE1) analog and PGE2 receptor agonist, has shown protection against cerebral ischemia. In this study, we tested the efficacy of misoprostol in the 12-month-old mice subjected to 1 of 2 complementary ICH models, the collagenase model (primary study) and blood model (secondary study, performed in an independent laboratory). We also investigated its potential mechanism of action. Misoprostol posttreatment decreased brain lesion volume, edema, and brain atrophy and improved long-term functional outcomes. In the collagenase-induced ICH model, misoprostol decreased cellular inflammatory response; attenuated oxidative brain damage and gelatinolytic activity; and decreased high-mobility group box 1 (HMGB1) expression, Src kinase activity, and interleukin-1β expression without affecting cyclooxygenase-2 expression. Furthermore, HMGB1 inhibition with glycyrrhizin decreased Src kinase activity, gelatinolytic activity, neuronal death, and brain lesion volume. Src kinase inhibition with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) decreased gelatinolytic activity and brain edema and improved neurologic function but did not decrease HMGB1 protein level. These results indicate that misoprostol protects brain against ICH injury through mechanisms that may involve the HMGB1, Src kinase, and matrix metalloproteinase-2/9 pathways.

Does Dabigatran Increase the Risk of Delayed Hematoma Expansion in a Rat Model of Collagenase-induced Intracerebral Hemorrhage?

Delayed hematoma expansion is common in intracerebral hemorrhage (ICH) patients using warfarin. Dabigatran induces fewer hemorrhagic complications compared with warfarin. However, the natural history of dabigatran-related ICH remains unclear. This study aims to clarify whether dabigatran increases the risk of delayed hematoma expansion in a rat ICH model. Male Wistar rats were treated with 2 dosages of dabigatran etexilate (DE: 10 mg/kg, n = 4; 20 mg/kg, n = 3) 30 minutes before ICH induction using intraparenchymal collagenase infusion. Five rats that received saline were used as controls. Magnetic resonance imaging was performed 24 and 48 hours after ICH induction, and serial hematoma volume measurements were obtained using T2-weighted images. Expanded hematoma volumes were calculated by subtracting hematoma volumes at 48 hours from those at 24 hours; the hematoma expansion rate was defined as the ratio of the expanded hematoma volume to that at 24 hours. The mean hematoma volumes (mm(3)) at 24 hours were 13.3 ± 3.3 in the control group, 14.9 ± 2.0 in the 10 mg/kg DE group, and 18.9 ± 7.6 in the 20 mg/kg DE group with no significant intergroup differences (P = .26). The mean hematoma volumes at 48 hours (mm(3)) were 21.7 ± 4.9 in the control group, 22.1 ± 5.0 in the 10 mg/kg DE group, and 23.4 ± 5.8 in the 20 mg/kg DE group with no significant intergroup differences (P = .90). Consequently, there were no significant intergroup differences in the hematoma expansion rates (P = .33). This experimental study of a rat ICH model indicates that dabigatran-related ICH may not increase the risk of delayed hematoma expansion.

MRI-based analysis of intracerebral hemorrhage in mice reveals relationship between hematoma expansion and the severity of symptoms.

Intracerebral hemorrhage (ICH) is featured by poor prognosis such as high mortality rate and severe neurological dysfunction. In humans, several valuables including hematoma volume and ventricular expansion of hemorrhage are known to correlate with the extent of mortality and neurological dysfunction. However, relationship between hematoma conditions and the severity of symptoms in animal ICH models has not been clarified. Here we addressed this issue by using 7-tesla magnetic resonance imaging (MRI) on collagenase-induced ICH model in mice. We found that the mortality rate and the performance in behavioral tests did not correlate well with the volume of hematoma. In contrast, when hemorrhage invaded the internal capsule, mice exhibited high mortality and showed poor sensorimotor performance. High mortality rate and poor performance in behavioral tests were also observed when hemorrhage invaded the lateral ventricle, although worsened symptoms associated with ventricular hemorrhage were apparent only during early phase of the disease. These results clearly indicate that invasion of the internal capsule or the lateral ventricle by hematoma is a critical determinant of poor prognosis in experimental ICH model in mice as well as in human ICH patients. MRI assessment may be a powerful tool to refine investigations of pathogenic mechanisms and evaluations of drug effects in animal models of ICH.

Hydrogen Inhalation Ameliorated Mast Cell–Mediated Brain Injury After Intracerebral Hemorrhage in Mice

Hydrogen inhalation was neuroprotective in several brain injury models. Its mechanisms are believed to be related to antioxidative stress. We investigated the potential neurovascular protective effect of hydrogen inhalation especially effect on mast cell activation in a mouse model of intracerebral hemorrhage. Controlled in vivo laboratory study. Animal research laboratory. One hundred seventy-one 8-week-old male CD-1 mice were used. Collagenase-induced intracerebral hemorrhage model in 8-week-old male CD-1 mice was used. Hydrogen was administrated via spontaneous inhalation. The blood-brain barrier permeability and neurologic deficits were investigated at 24 and 72 hours after intracerebral hemorrhage. Mast cell activation was evaluated by Western blot and immuno-staining. The effects of hydrogen inhalation on mast cell activation were confirmed in an autologous blood injection model intracerebral hemorrhage. At 24 and 72 hours post intracerebral hemorrhage, animals showed blood-brain barrier disruption, brain edema, and neurologic deficits, accompanied with phosphorylation of Lyn kinase and release of tryptase, indicating mast cell activation. Hydrogen treatment diminished phosphorylation of Lyn kinase and release of tryptase, decreased accumulation and degranulation of mast cells, attenuated blood-brain barrier disruption, and improved neurobehavioral function. Activation of mast cells following intracerebral hemorrhage contributed to increase of blood-brain barrier permeability and brain edema. Hydrogen inhalation preserved blood-brain barrier disruption by prevention of mast cell activation after intracerebral hemorrhage.

Ethyl pyruvate ameliorates intracerebral hemorrhage-induced brain injury through anti-cell death and anti-inflammatory mechanisms

Ethyl pyruvate (EP) is a pyruvate derivative and known to be cytoprotective in various pathological conditions through anti-cell death and anti-inflammatory mechanisms. The present study investigated the neuroprotective effect of ethyl pyruvate using a mouse model of collagenase-induced intracerebral hemorrhage (ICH). Our results showed that EP treatment to mice reduced brain edema and improved neurological function after ICH. Delayed treatment with EP until 6h after ICH to mice was still neuroprotective. We further demonstrated that EP protected neurons from hemoglobin-induced cell death in vitro and neuronal cell degeneration in ICH mice. Moreover, EP exerted anti-inflammatory effects by inhibiting microglia activation, nuclear factor-κB (NF-κB) DNA binding activity and subsequent downstream pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)) production. Taken together, these results suggest that EP exerts neuroprotective effect via anti-cell death and anti-inflammatory actions. EP is a potential novel treatment for ICH patients and deserves further investigation.

Intravenous tPA Therapy Does Not Worsen Acute Intracerebral Hemorrhage in Mice

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for reperfusing ischemic strokes. But widespread use of tPA is still limited by fears of inadvertently administering tPA in patients with intracerebral hemorrhage (ICH). Surprisingly, however, the assumption that tPA will worsen ICH has never been biologically tested. Here, we assessed the effects of tPA in two models of ICH. In a mouse model of collagenase-induced ICH, hemorrhage volumes and neurological deficits after 24 hrs were similar in saline controls and tPA-treated mice, whereas heparin-treated mice had 3-fold larger hematomas. In a model of laser-induced vessel rupture, tPA also did not worsen hemorrhage volumes, while heparin did. tPA is known to worsen neurovascular injury by amplifying matrix metalloproteinases during cerebral ischemia. In contrast, tPA did not upregulate matrix metalloproteinases in our mouse ICH models. In summary, our experimental data do not support the assumption that intravenous tPA has a deleterious effect in acute ICH. However, due to potential species differences and the inability of models to fully capture the dynamics of human ICH, caution is warranted when considering the implications of these findings for human therapy.

Activation of cerebral recovery by matrix metalloproteinase-9 after intracerebral hemorrhage

Cerebral ischemia, traumatic brain injury, intracerebral hemorrhage and other brain insults trigger neurogenesis in the subventricular zone and hippocampal subgranular zone, and newly formed blood vessels promote the migration of these new neuronal cells to damaged brain regions. The molecular steps involved in brain injury-induced angiogenesis and neurogenesis are unclear. Here we used a rat model of collagenase-induced intracerebral hemorrhage (ICH) to examine whether matrix metalloproteinase-9 (MMP-9), a zinc endopeptidase that regulates growth factor levels during recovery from brain injury, is involved in neurogenesis and angiogenesis following ICH. Induction of ICH led to significant increases in the levels of MMP-9, vascular endothelial growth factor (VEGF), and nerve growth factor (NGF), as well as in the numbers of 5-bromo-2-deoxyuridine (BrdU)- and doublecortin (DCX)-positive cells, in the ipsilateral brain. Intracerebroventricular injection of MMP-9 siRNA reduced these ICH-induced increases. These findings suggest that MMP-9 may promote angiogenesis and neurogenesis during recovery from ICH.

Effects of high-mobility group box1 on cerebral angiogenesis and neurogenesis after intracerebral hemorrhage

Neural stem cells, which reside mainly in the subventricular and subgranular zones of the hippocampus, can regenerate new neuroblasts after various brain insults. Aided by vascular remodeling, these new neuroblasts migrate long distances to injured brain regions. Studies have suggested that high-mobility group box1 (HMGB1), a nonhistone nuclear DNA-binding protein, may stimulate such remodeling in the late phase of some types of brain injury, but it is unclear whether this is true for intracerebral hemorrhage (ICH). Here we used a rat model of collagenase-induced ICH to determine whether HMGB1 can promote neurogenesis and angiogenesis in the late phase of injury. Daily administration of ethyl pyruvate, which inhibited HMGB1 expression, reduced the recovery of neurological function, decreased vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) levels in the ipsilateral striatum, and decreased the numbers of 5-bromo-2-deoxyuridine (BrdU)- and doublecortin (DCX)-positive cells around the hematoma. These findings suggest that HMGB1 may promote angiogenesis and neurogenesis in the late phase of ICH.

High-mobility group box1 protein promotes neuroinflammation after intracerebral hemorrhage in rats

High-mobility group box1 (HMGB1) protein is massively released into the cytoplasm and induces inflammation following various insults such as sepsis, acute cerebral ischemia, and pancreatitis. However, whether HMGB1 can act as an early proinflammatory cytokine to promote inflammation after intracerebral hemorrhage (ICH) is unclear. We explored this question using a rat model of collagenase-induced ICH. We found that HMGB1 was released into the cytoplasm soon after ICH. Administration of ethyl pyruvate decreased the level of HMGB1 and microglia around the hematoma. Ethyl pyruvate also ameliorated ICH-induced neuronal apoptosis, cerebral edema, and neurological impairment. These findings suggest that HMGB1 may act as an early proinflammatory cytokine within the neurovascular unit to mediate inflammation during the acute phase of ICH.

Astrocyte-Specific Expression of Survivin after Intracerebral Hemorrhage in Mice: A Possible Role in Reactive Gliosis?

Intracerebral hemorrhage (ICH), the most common form of hemorrhagic stroke, accounts for up to 15% of all strokes. Despite maximal surgical intervention and supportive care, ICH is associated with significant morbidity and mortality, in part due to a lack of viable treatment options. Astrogliosis, a key feature of secondary injury that is characterized by glial proliferation, is a poorly-defined process that may produce both beneficial and detrimental outcomes after brain injury. Using a pre-clinical murine model of collagenase-induced ICH, we demonstrate a delayed upregulation of survivin, a key molecule involved in tumor cell proliferation and survival, by 72 h post-ICH. Notably, this increase in survivin expression was prominent in GFAP-positive astrocytes, but absent in neurons. Survivin was not expressed at detectable levels in the striatum of sham-operated mice. The expression of survivin after ICH was temporally and spatially associated with the expression of proliferating cell nuclear antigen (PCNA), an established marker of cellular proliferation. Moreover, the survivin expression was co-localized in proliferating astrocytes as evidenced by triple-label immunohistochemistry. Finally, shRNA-mediated silencing of survivin expression attenuated PCNA expression and reduced cellular proliferation in human glial cells. Together, these data suggest a potentially novel role for survivin in functionally promoting astrocytic proliferation after ICH.

Caveolin-1 Deletion Reduces Early Brain Injury after Experimental Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a subtype of stroke with high rates of morbidity and mortality. Caveolin-1 (Cav-1) is the main structural protein of caveolae and is involved in regulating signal transduction and cholesterol trafficking in cells. Although a recent study suggests a protective role of Cav-1 in cerebral ischemia, its function in ICH remains unknown. In this study, we examined the role of Cav-1 and in a model of collagenase-induced ICH and in neuronal cultures. Our results indicate that Cav-1 was up-regulated in the perihematomal area predominantly in endothelial cells. Cav-1 knockout mice had smaller injury volumes, milder neurologic deficits, less brain edema, and neuronal death 1 day after ICH than wild-type mice. The protective mechanism in Cav-1 knockout mice was associated with marked reduction in leukocyte infiltration, decreased expression of inflammatory mediators, including macrophage inflammatory protein (MIP)-2 and cyclooxygenase (COX)-2, and reduced matrix metalloproteinase-9 activity. Deletion of Cav-1 also suppressed heme oxygenase-1 expression and attenuated reactive oxygen species production after ICH. Moreover, deletion or knockdown of Cav-1 decreased neuronal vulnerability to hemin-induced toxicity and reduced heme oxygenase (HO)-1 induction in vitro. These data suggest that Cav-1 plays a deleterious role in early brain injury after ICH. Inhibition of Cav-1 may provide a novel therapeutic approach for the treatment of hemorrhagic stroke.

Serum S100B, brain edema, and hematoma formation in a rat model of collagenase-induced hemorrhagic stroke

S100B, a 21-kD Ca(2+) binding protein expressed in Schwann cells and astroglia, has often been reported as a promising biomarker for ischemic stroke. In addition to ischemic stroke, the peripheral S100B level may also be useful as a biomarker for intracerebral hemorrhage (ICH). However, the kinetics and characterization of peripheral S100B in patients or experimental animal models with ICH have not been carefully examined. The present study investigated the kinetics and characteristics of the serum S100B level in a rat collagenase-induced ICH model. The serum S100B kinetics and the time-course of brain edema and hematoma formation were examined. Then, the correlations between the elevated serum S100B level and brain edema or hematoma formation were investigated. A transient elevation of serum S100B that peaked at 6 h after ICH induction was observed. The single measurement of serum S100B at 6 h after ICH induction was significantly correlated with brain edema formation and the maximal extent of the hematoma volumes. These results suggest the significance of serum S100B as a biomarker of brain damage resulting from ICH.

Heme oxygenase 2 deficiency increases brain swelling and inflammation after intracerebral hemorrhage

Intracerebral hemorrhage (ICH) remains a major medical problem and currently has no effective treatment. Hemorrhaged blood is highly toxic to the brain, and catabolism of the pro-oxidant heme, mainly released from hemoglobin, is critical for the resolution of hematoma after ICH. The degradation of the pro-oxidant heme is controlled by heme oxygenase (HO). We have previously reported a neuroprotective role for HO2 in early brain injury after ICH; however, in vivo data that specifically address the role of HO2 in brain edema and neuroinflammation after ICH are absent. Here, we tested the hypothesis that HO2 deletion would exacerbate ICH-induced brain edema, neuroinflammation, and oxidative damage. We subjected wild-type (WT) and HO2 knockout ( −/−) mice to the collagenase-induced ICH model. Interestingly, HO2 −/− mice had enhanced brain swelling and neuronal death, although HO2 deletion did not increase collagenase-induced bleeding; the exacerbation of brain injury in HO2 −/− mice was also associated with increases in neutrophil infiltration, microglial/macrophage and astrocyte activation, DNA damage, peroxynitrite production, and cytochrome c immunoreactivity. In addition, we found that hemispheric enlargement was more sensitive than brain water content in the detection of subtle changes in brain edema formation in this model. Combined, these novel findings extend our previous observations and demonstrate that HO2 deficiency increases brain swelling, neuroinflammation, and oxidative damage. The results provide additional evidence that HO2 plays a critical protective role against ICH-induced early brain injury.

Evolution of the inflammatory response in the brain following intracerebral hemorrhage and effects of delayed minocycline treatment

There are no effective treatments for intracerebral hemorrhage (ICH). Although inflammation is a potential therapeutic target, there is a dearth of information about time-dependent and cell-specific changes in the expression of inflammation-related genes. Using the collagenase-induced ICH model in rats and real-time quantitative RT-PCR we monitored mRNA levels of markers of glial activation, pro- and anti-inflammatory cytokines, enzymes responsible for cytokine activation and several matrix metalloproteases at 6 h and 1, 3 and 7 days after ICH onset. For the most highly up-regulated genes, immunohistochemistry was then used to identify cell-specific protein expression. Finally, minocycline, a drug widely reported to reduce damage in several models of brain injury, was used to test the hypothesis that it can reduce up-regulation of inflammation-related genes when administered using a clinically relevant dosing regime: intraperitoneal injection beginning 6 h after ICH. Our results show a complex inflammatory response, with different brain cell types producing several pro- and anti-inflammatory molecules for at least 7 days after ICH onset. Included is the first demonstration that astrocytes are an important source of interleukin-1beta (IL-1beta), interleukin-1 receptor antagonist (IL-1ra), interleukin-6 (IL-6) and MMP-12. Importantly, our results demonstrate that while delayed minocycline treatment effectively reduces early up-regulation of TNFalpha and MMP-12, its efficacy is lost when treatment is extended for up to a week, and it does not reduce several other genes associated with microglia activation. These results suggest caution in extrapolating to ICH the promising results of minocycline treatment in other models of brain injury.