G. Xi

Blood-brain barrier function in intracerebral hemorrhage

In this paper, we review current knowledge on blood-brain barrier (BBB) dysfunction following intracerebral hemorrhage (ICH). BBB disruption is a hallmark of ICH-induced brain injury. Such disruption contributes to edema formation, the influx of leukocytes, and the entry of potentially neuroactive agents into the perihematomal brain, all of which may contribute to brain injury. A range of factors have been implicated in inducing BBB disruption, including inflammatory mediators (e.g., cytokines and chemokines), thrombin, hemoglobin breakdown products, oxidative stress, complement, and matrix metalloproteinases. While there is interaction between some of these mediators, it is probable that prevention of ICH-induced BBB disruption will involve blocking multiple pathways or blocking a common end pathway (e.g., by stabilizing tight junction structure). While the effects of ICH on BBB passive permeability have been extensively examined, effects on other barrier properties (metabolic and transport functions) have been less well-studied. However, recent data suggests that ICH can affect transport and that this may help protect the BBB and the brain. Indeed, it is possible in small bleeds that BBB disruption may be beneficial, and it is only in the presence of larger bleeds that disruption has detrimental effects.

Microglial activation and brain injury after intracerebral hemorrhage

Microglial activation and thrombin formation contribute to brain injury after intracerebral hemorrhage (ICH). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) are 2 major proinflammatory cytokines. In this study, we investigated whether thrombin stimulates TNF-alpha and IL-1beta secretion in vitro, and whether microglial inhibition reduces ICH-induced brain injury in vivo. There were 2 parts to this study. In the first part, cultured rat microglial cells were treated with vehicle, thrombin (5 and 10U/mL), or thrombin plus tuftsin (0.05 microg/mL), an inhibitor of microglia activation. Levels of TNF-alpha and IL-1beta in culture medium were measured by ELISA at 4, 8, and 24 h after thrombin treatment. In the second part of the study, rats received an intracerebral infusion of 100 microL autologous whole blood with or without 25 microg of tuftsin 1-3 fragment. Rats were killed at day 1 or day 3 for immunohistochemistry and brain water content measurement. We found that thrombin receptors were expressed in cultured microglia cells, and TNF-alpha and IL-1beta levels in the culture medium were increased after thrombin treatment. Tuftsin reduced thrombin-induced upregulation of TNF-alpha and IL-1beta. In vivo, microglia were activated after ICH, and intracerebral injection of tuftsin reduced brain edema in the ipsilateral basal ganglia (81.1 +/- 0.7% vs. 82.7 +/- 1.3% in vehicle-treated group; p < 0.05) after ICH. These results suggest a critical role of microglia activation in ICH-related brain injury.

Deferoxamine reduces CSF free iron levels following intracerebral hemorrhage

Iron overload occurs in brain after intracerebral hemorrhage (ICH). Deferoxamine, an iron chelator, attenuates perihematomal edema and oxidative stress in brain after ICH. We investigated the effects of deferoxamine on cerebrospinal fluid (CSF) free iron and brain total iron following ICH. Rats received an infusion of 100-microL autologous whole blood into the right basal ganglia, then were treated with either deferoxamine (100 mg/kg, i.p., administered 2 hours after ICH and then at 12-hour intervals for up to 7 days) or vehicle. The rats were killed at different time points from 1 to 28 days for measurement of free and total iron. Behavioral tests were also performed. Free iron levels in normal rat CSF were very low (1.1 +/- 0.4 micromol). After ICH, CSF free iron levels were increased at all time points. Levels of brain total iron were also increased after ICH (p < 0.05). Deferoxamine given 2 hours after ICH reduced free iron in CSF at all time points. Deferoxamine also reduced ICH-induced neurological deficits (p < 0.05), but did not reduce total brain iron. In conclusion, CSF free iron levels increase after ICH and do not clear for at least 28 days. Deferoxamine reduces free iron levels and improves functional outcome in the rat, indicating that it may be a potential therapeutic agent for ICH patients.

Iron-induced oxidative brain injury after experimental intracerebral hemorrhage

We investigated the occurrence of DNA damage in brain after intracerebral hemorrhage (ICH) and the role of iron in such injury. Male Sprague-Dawley rats received an infusion of 100 microL autologous whole blood or 30 microL FeCl2 into the right basal ganglia and were sacrificed 1, 3, or 7 days later. 8-hydroxyl-2-deoxyguanosine (8-OHdG) was analyzed by immunohistochemistry, while the number of apurinic/apyrimidinic abasic sites (AP sites) was also quantified. 8-OHdG and AP sites are two hallmarks of DNA oxidation. DNA damage was also examined using PANT and TUNEL labeling. Dinitrophenyl (DNP) was measured by Western blot to compare the time course of protein oxidative damage to that of DNA. DNA repair APE/Ref-1 and Ku-proteins were also measured by Western blot. Bipyridine, a ferrous iron chelator, was used to examine the role of iron in ICH-induced oxidative brain injury. An increase in 8-OHdG, AP sites, and DNP levels, and a decrease in APE/Ref-1 and Ku levels were observed. Abundant PANT-positive cells were also observed in the perihematomal area 3 days after ICH. Bipyridine attenuated ICH-induced changes in PANT and DNP. These results suggest that iron-induced oxidation causes DNA damage in brain after ICH and that iron is a therapeutic target for ICH.

Systemic zinc protoporphyrin administration reduces intracerebral hemorrhage-induced brain injury

Hemoglobin degradation products result in brain injury after intracerebral hemorrhage (ICH). Recent studies found that intracerebral infusion of heme oxygenase inhibitors reduces hemoglobin- and ICH-induced brain edema in rats and pigs. The present study examined whether systemic use of zinc protoporphyrin (ZnPP), a heme oxygenase inhibitor, can attenuate brain edema, behavioral deficits, and brain atrophy following ICH. All rats had intracerebral infusion of 100-microL autologous blood. ZnPP (1 nmol/hour/rat) or vehicle was given immediately or 6 hours following ICH. ZnPP was delivered intraperitoneally up to 14 days through an osmotic mini-pump. Rats were killed at day 3 and day 28 after ICH for brain edema and brain atrophy measurements, respectively. Behavioral tests were performed. We found that ZnPP attenuated brain edema in animals sacrificed 3 days after ICH (p < 0.05). ZnPP also reduced ICH-induced caudate atrophy (p < 0.05) and ventricular enlargement (p < 0.05). In addition, ZnPP given immediately or 6 hours after ICH improved neurological deficits (p < 0.05). In conclusion, systemic zinc protoporphyrin treatment started at 0 or 6 hours after ICH reduced brain edema, neurological deficits, and brain atrophy after ICH. These results indicate that heme oxygenase may be a new target for ICH therapeutics.

Brain edema from intracerebral hemorrhage.

Sequential changes in brain parenchyma surrounding an intracerebral hemorrhage are described here. Re-bleeding occurs within the first several hours after the initial hemorrhage in about 30%, of cases. The coagulation cascade is activated as soon as blood encounters tissue. Perihematomal brain edema develops in response to clot retraction, thrombin formation, erythrocyte lysis, hemoglobin toxicity, complement activation, mass effect, and blood-brain barrier disruption. Early hematoma evacuation interrupts edema formation. The toxicity ofextravasated blood in brain parenchyma has not been studied well in traumatic injury or in hemorrhagic tumor models yet, but similar mechanisms of edema formation are likely to occur in these conditions.

Effects of endogenous and exogenous estrogen on intracerebral hemorrhage-induced brain damage in rats

The present study examined differences in intracerebral hemorrhage (ICH)-induced brain injury in male and female rats, whether delayed administration of 17beta-estradiol can reduce ICH-induced brain damage, and whether these effects are estrogen receptor (ER)-dependent. Male and female Sprague-Dawley rats received an infusion of 100-microL autologous whole blood into the right basal ganglia. The effects of 1beta-estradiol (5 mg/kg, i.p.) on ICH-induced brain injury were examined by measuring brain edema and neurological deficits 24 hours later. Heme oxygenase-1 (HO-1) was investigated by immuno-analysis. Brain edema was significantly less in female compared to male rats. The ER antagonist ICI182,780 exacerbated ICH-induced brain edema in female but not in male rats, suggesting that ER activation during ICH is protective in female rats. Administration of 17beta-estradiol to male (but not female) rats significantly reduced brain edema, neurological deficits, and ICH-induced increases in brain HO-1 levels when given 2 hours after ICH. This study showed that female rats have less ICH-induced injury than male rats. ER is involved in limiting ICH-induced injury in female rats. ICH-injury in male rats can be reduced by 17beta-estradiol. Since 17beta-estradiol treatment was effective in male rats, it could be a potential therapeutic agent for ICH.

Deferoxamine therapy for intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is a subtype of stroke with very high mortality. Experiments have indicated that clot lysis and iron play an important role in ICH-induced brain injury. Iron overload occurs in the brain after ICH in rats. Intracerebral infusion of iron causes brain edema and neuronal death. Deferoxamine, an iron chelator, is an FDA-approved drug for the treatment of acute iron intoxication and chronic iron overload due to transfusion-dependent anemia. Deferoxamine can rapidly penetrate the blood-brain barrier and accumulate in the brain tissue in significant concentration after systemic administration. We have demonstrated that deferoxamine reduces ICH-induced brain edema, neuronal death, brain atrophy, and neurological deficits. Iron chelation with deferoxamine could be a new therapy for ICH.

Spontaneous intracerebral hemorrhage in humans: hematoma enlargement, clot lysis, and brain edema

Early hematoma enlargement and delayed clot lysis contribute to brain injury after intracerebral hemorrhage (ICH). We investigated hematoma growth, clot lysis, and brain edema formation in patients with spontaneous ICH. A total of 17 spontaneous ICH patients who received regular medication were chosen for this study. All patients had their first CT scan within 5 hours of onset of symptoms (day 0). The patients then underwent second, third, and fourth CT scans at 1, 3, and 10 days later. Hematoma size and absolute and relative brain edema volumes were measured. Hematoma enlargement was defined as a > 33% increase in volume. Relative brain edema volume = absolute brain edema volume/hematoma size. Hematoma enlargement occurred in 4 of the 17 ICH patients (24%) within the first 24 hours. The hematoma sizes were reduced significantly at day 10 (p < 0.05) because of clot lysis. However, both absolute and relative brain edema increased gradually with time (p < 0.01). These results suggest that delayed brain edema following ICH may result from hematoma lysis. This study also shows that early hematoma enlargement occurs in Chinese patients with ICH. Reducing early hematoma growth and limiting clot lysis-induced brain toxicity could be potential therapies for ICH.

A thrombin inhibitor reduces brain edema, glioma mass and neurological deficits in a rat glioma model

Although thrombin is a critical enzyme in the coagulation cascade, it has become apparent that it has many other effects. Thus, it may induce brain edema formation, angiogenesis and cell proliferation. Because of the importance of these three factors in the extremely poor prognosis of glioma patients, the present study examined the role of thrombin in that disease state. We found that thrombin activity is increased in a rat glioma model and thrombin positive cells were present in the tumor. Anti-thrombin treatment with argatroban reduced brain edema, tumor growth, and tumor-related neurological deficits. Our results suggest that thrombin is a new target for glioma treatment.