Guohua Xi

Mechanisms of brain injury after intracerebral haemorrhage

The past decade has resulted in a rapid increase in knowledge of mechanisms underlying brain injury induced by intracerebral haemorrhage (ICH). Animal studies have suggested roles for clot-derived factors and the initial physical trauma and mass effect as a result of haemorrhage. The coagulation cascade (especially thrombin), haemoglobin breakdown products, and inflammation all play a part in ICH-induced injury and could provide new therapeutic targets. Human imaging has shown that many ICH continue to expand after the initial ictus. Rebleeding soon after the initial haemorrhage is common and forms the basis of a current clinical trial using factor VIIa to prevent rebleeding. However, questions about mechanisms of injuries remain. There are conflicting data on the role of ischaemia in ICH and there is uncertainty over the role of clot removal in ICH therapy. The next decade should bring further information about the underlying mechanisms of ICH-induced brain injury and new therapeutic interventions for this severe form of stroke. This review addresses our current understanding of the mechanisms underlying ICH-induced brain injury.

Intracerebral haemorrhage: mechanisms of injury and therapeutic targets

Intracerebral haemorrhage accounts for about 10-15% of all strokes and is associated with high mortality and morbidity. No successful phase 3 clinical trials for this disorder have been completed. In the past 6 years, the number of preclinical and clinical studies focused on intracerebral haemorrhage has risen. Important advances have been made in animal models of this disorder and in our understanding of mechanisms underlying brain injury after haemorrhage. Several therapeutic targets have subsequently been identified that are now being pursued in clinical trials. Many clinical trials have been based on limited preclinical data, and guidelines to justify taking preclinical results to the clinic are needed.

Behavioral Tests After Intracerebral Hemorrhage in the Rat

Background and Purpose— In humans, intracerebral hemorrhage (ICH) causes marked perihematomal edema formation and neurological deficits. A rat ICH model, involving infusion of autologous blood into the caudate, has been used extensively to study mechanisms of edema formation, but an examination of behavioral outcome would improve its preclinical utility and provide a more rigorous assessment of the pathological cascade of events over time. The purpose of this study was to use a battery of sensorimotor function tests to examine the neurological effects of ICH in the rat and to examine which components of the hematoma are involved in generating those effects. Methods— The behavioral tests used were forelimb placing, preference for forelimb use for weight shifts during vertical exploration of a cylindrical enclosure, and a corner turn test. Rats were tested from day 1 to day 28 after injection of autologous whole blood; injection of blood plus hirudin (thrombin inhibitor), packed red blood cells, thrombin, or saline; or needle placement only. Results— The battery of tests indicated that there were marked neurological deficits by day 1 after ICH, with progressive recovery of function over 4 weeks. The forelimb placing score paralleled changes in edema. Injection of thrombin caused and injection of hirudin reduced the ICH-induced neurological deficits. Injection of packed red blood cells, which causes delayed edema formation, induced delayed neurological deficits Conclusions— These tests allow continuous monitoring of neurological deficits after rat ICH and assessment of therapeutic interventions. The time course of the neurological deficit closely matched the time course of cerebral edema for both ICH and injection of blood components. There was marked recovery of function after ICH, which may be amenable to therapeutic manipulation.

Iron and Iron-Handling Proteins in the Brain After Intracerebral Hemorrhage

Background and Purpose— Evidence indicates that brain injury after intracerebral hemorrhage (ICH) is due in part to the release of iron from hemoglobin. Therefore, we examined whether such iron is cleared from the brain and the effects of ICH on proteins that may alter iron release or handling: brain heme oxygenase-1, transferrin, transferrin receptor, and ferritin. Methods— Male Sprague-Dawley rats received an infusion of 100 &mgr;L autologous whole blood into the right basal ganglia and were killed 1, 3, 7, 14, or 28 days later. Enhanced Perl’s reaction was used for iron staining, and brain nonheme iron content was determined. Brain heme oxygenase-1, transferrin, transferrin receptor, and ferritin were examined by Western blot analysis and immunohistochemistry. Immunofluorescent double labeling was performed to identify which cell types express ferritin. Results— ICH upregulated heme oxygenase-1 levels and resulted in iron overload in the brain. A marked increase in brain nonheme iron was not cleared within 4 weeks. Brain transferrin and transferrin receptor levels were also increased. In addition, an upregulation of ICH on ferritin was of very long duration. Conclusions— The iron overload and upregulation of iron-handling proteins, including transferrin, transferrin receptor, and ferritin, in the brain after ICH suggest that iron could be a target for ICH therapy.

The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury : deleterious or protective?

In the last two decades it has become apparent that thrombin has many extravascular effects that are mediated by a family of protease‐activated receptors (PARs). PAR‐1, ‐3 and ‐4 are activated via cleavage by thrombin. The importance of extravascular thrombin in modulating ischemic, hemorrhagic and traumatic injury in brain has recently become clear. Thus, in vitro, thrombin at low concentration protects neurons and astrocytes from cell death caused by a number of different insults. In vivo, pretreating the brain with a low dose of thrombin (thrombin preconditioning), attenuates the brain injury induced by a large dose of thrombin, an intracerebral hemorrhage or by focal cerebral ischemia. Thrombin may also be an important mediator of ischemic preconditioning. In contrast, high doses of thrombin kill neurons and astrocytes in vitro and cause disruption of the blood–brain barrier, brain edema and seizures in vivo. This review examines the role of thrombin in brain injury and the molecular mechanisms and signaling cascades involved.

Brain Injury After Intracerebral Hemorrhage: The Role of Thrombin and Iron

Intracerebral hemorrhage (ICH) is a subtype of stroke with high morbidity and mortality. The mechanisms underlying ICH-induced brain injury have become better understood during the past decade. Experimental investigations have indicated that thrombin formation, red blood cell lysis, and iron toxicity play a major role in ICH-induced injury and that these mechanisms may provide new therapeutic targets. This article reviews the role of thrombin and iron in ICH-induced injury.

Attenuation of Thrombin-Induced Brain Edema by Cerebral Thrombin Preconditioning

BACKGROUND AND PURPOSE Edema formation after intracerebral hemorrhage has been linked to thrombin toxicity induced by the clot. However, thrombin at low concentrations actually protects neurons and astrocytes in culture from hypoglycemic and ischemic cell death. It is also known that a brief episode of brain ischemia increases neuronal tolerance to a subsequent severe ischemic episode. The objective of this study was to investigate whether pretreatment of the brain with low-dose thrombin induces tolerance to a subsequent large dose of thrombin injected into brain parenchyma. METHODS The rat brain was preconditioned with 1 U thrombin by direct infusion into the right caudate nucleus. After thrombin pretreatment, the effects of a large dose (5 U) of thrombin on brain edema formation were studied at different intervals. We examined whether heat-shock protein (HSP) 27, HSP32, and HSP70 were induced by Western blot analysis, immunocytochemistry, and immunofluorescent double staining. RESULTS Thrombin pretreatment significantly attenuated the brain edema that normally follows the infusion of a large dose of thrombin (79.2+/-0.4 versus 84.0+/-0.3; P<0.01). This effect was abolished by the thrombin inhibitor hirudin. Time course studies showed that the maximal effect of thrombin preconditioning (TPC) on brain edema formation was 7 days after pretreatment. This time course corresponded to marked upregulation of HSP27 in the ipsilateral brain. TPC also induced HSP32, but this effect occurred earlier than the effect on edema formation. TPC had no effect on HSP70. Immunocytochemistry and immunofluorescent double labeling showed that HSP27 and HSP32 were expressed in astrocytes after TPC. CONCLUSIONS OFF phenomenon of thrombin-induced tolerance of the brain to edema formation may be related to HSP27 induction.

Oxidative brain injury from extravasated erythrocytes after intracerebral hemorrhage

Intracerebral infusion of lysed erythrocytes causes brain edema without inducing ischemic cerebral blood flow. Reports have indicated that oxidative damage contributes to secondary brain injury in stroke. In the present study, we investigated whether erythrocyte lysis after intracerebral hemorrhage (ICH) might result in oxidative brain damage. This study had four parts. Male Sprague-Dawley rats received an infusion of autologous lysed erythrocytes into the right striatum. Control rats only had a needle insertion. Neurological deficits, brain water and ion contents were determined in the first part. In the second part, hemoxygenase-1 (HO-1), manganese superoxide dismutase (Mn-SOD), copper/zinc SOD (CuZn-SOD) and protein carbonyl levels were determined by Western blot analysis. In the third part, immunohistochemistry was performed for HO-1. DNA damage was examined using DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) in the fourth part. Infusion of lysed RBCs induced marked edema in the ipsilateral striatum and profound neurological deficits. Western blot analysis and immunohistochemistry indicated that HO-1 was upregulated 24 h after infusion of lysed red blood cells. Both Mn-SOD and CuZn-SOD contents decreased, protein carbonyl levels increased in the ipsilateral striatum, and there was the appearance of PANT- and TUNEL-positive cells suggesting oxidative mechanisms in the erythrocyte-induced brain injury. In conclusion, oxidative stress caused by components of the lysed erythrocytes contributes to the brain injury after ICH.

Pathophysiology of brain edema formation.

A number of mechanisms seem to be involved in edema formation after an ICH. At least three phases of edema are involved in ICH. These include a very early phase (first several hours) involving hydrostatic pressure and clot retraction, a second phase (first 2 days) involving the activation of the coagulation cascade and thrombin production, and a third phase (after 3 days) involving RBC lysis and hemoglobin-induced neuronal toxicity. Activation of the complement system in brain parenchyma also plays an important role in the second and third phases. There are potential therapeutic strategies to address each of these mechanisms. Because the adverse effect of an ICH seems to result from a toxic effect of blood components on brain tissue, early clot removal may be the best strategy, because it results in the removal of all the toxic components [93]. Hematoma aspiration after tissue plasminogen activator (tPA) infusion has also been shown to be relatively safe and effective in animal models. Kaufman et al [94] reported that tPA lysed the hematoma in minutes and did not cause inflammation or bleeding in rabbits. Because clots lysed with tPA can be aspirated through a needle or catheter, mechanical brain injury by this method is minimized. In a rat model, aspiration of clot with tPA reduced clot volume and brain injury [95,96]. Recently, Wagner et al [97] infused tPA into hematomas in a porcine model at 3 hours after induction and aspirated the liquified clots 1 hour later. Clot removal after tPA treatment resulted in a 72% reduction in hematoma volume compared with untreated controls. Clot removal also reduced brain edema volume and BBB disruption and improved cerebral tissue pressure [93]. Six randomized trials have been accomplished, but surgical evacuation of the clot remains controversial [98-103]. Recently, thrombolysis and aspiration under CT guidance reduced the hematoma volume effectively [104]. Infusion of tPA directly into the hematoma before clot aspiration has also been used in human beings. Up to 90% of the original hematoma volume can be removed [105, 106]. Schaller et al [107] injected tPA directly into a hematoma 72 hours after the ictus in patients. The hematomas were lysed, and the liquified clots were drained in 14 patients. Two patients died, but none had recurrent hemorrhage. In conclusion, much has been learned about the basic mechanisms involved in edema formation after ICH. Animal models indicate that a number of components of blood are capable of inducing brain injury and brain edema. Now, it is time to translate that basic information into clinical trials.

Deferoxamine Reduces Intracerebral Hematoma-Induced Iron Accumulation and Neuronal Death in Piglets

Background and Purpose— Our previous studies found that deferoxamine reduces intracerebral hemorrhage (ICH)-induced brain injury in rats. The current study examined whether deferoxamine reduces brain injury in a piglet ICH model. Methods— Pigs received an injection of autologous blood into the right frontal lobe. Deferoxamine (50 mg/kg, IM) or vehicle was administered 2 hours after ICH and then every 12 hours up to 7 days. Animals were killed 3 or 7 days later to examine iron accumulation, white matter injury, and neuronal death. Results— ICH resulted in development of a reddish perihematomal zone, and iron accumulation, ferritin upregulation, and neuronal death within that zone. Deferoxamine reduced the perihematomal reddish zone, white matter injury, and the number of Perls’, ferritin, and Fluoro-Jade C–positive cells. Conclusions— Iron accumulation occurs in the piglet brain after ICH. Deferoxamine reduces ICH-induced iron buildup and brain injury in piglets.