Richard F. Keep

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.

Monocyte chemoattractant protein-1 regulation of blood-brain barrier permeability

The present study was designed to elucidate the effects of the chemokine monocyte chemoattractant protein (MCP-1) on blood–brain barrier (BBB) permeability. Experiments were conducted under in vitro conditions (coculture of brain endothelial cells and astrocytes) to study the cellular effects of MCP-1 and under in vivo conditions (intracerebral and intracerebroventricular administration of MCP-1) to study the potential contribution of MCP-1 to BBB disruption in vivo. Our results showed that MCP-1 induces a significant increase in the BBB permeability surface area product for fluorescein isothiocyanate (FITC)-albumin under in vivo conditions, particularly during prolonged (3 or 7 days) exposure (0.096±0.008 versus 0.031±0.005 μL/g min in controls at 3 days, P<0.001). Monocyte chemoattractant protein-1 also enhanced (17-fold compared with control) the permeability of the in vitro BBB (coculture) model. At the cellular level, MCP-1 causes alteration of tight junction (TJ) proteins in endothelial cells (redistribution of TJ proteins determined by Western blotting and loss of immunostaining for occludin, claudin-5, ZO-1, ZO-2). Monocyte chemoattractant protein-1-induced alterations in BBB permeability are mostly realized through the CCR2 receptor. Absence of CCR2 diminishes any effect of MCP-1 on BBB permeability in vitro and in vivo. The permeability surface area product for FITC-albumin after 3 days exposure to MCP-1 was 0.096±0.006 and 0.032±0.007 μL/g min, in CCR2+/+ and CCR2−/− mice, respectively (P<0.001). Monocytes/macrophages also participate in MCP-1-induced alterations in BBB permeability in vivo. Monocytes/macrophages depletion (by clodronate liposomes) reduced the effect of MCP-1 on BBB permeability in vivo ∼2 fold. Our results suggest that, besides its main function of recruiting leukocytes at sites of inflammation, MCP-1 also plays a role in ‘opening’ the BBB.

Potential role of MCP-1 in endothelial cell tight junction `opening': signaling via Rho and Rho kinase

The expression of the monocyte chemoattractant protein-1 (MCP-1) receptor CCR2 by brain endothelial cells suggests that MCP-1 may have other functions than purely driving leukocyte migration into brain parenchyma during inflammation. This study examines one of these potential novel roles of MCP-1 regulation of endothelial permeability using primary cultures of mouse brain endothelial cells. MCP-1 induces reorganization of actin cytoskeleton (stress fiber formation) and redistribution of tight junction proteins, ZO-1, ZO-2 occludin and claudin-5, from the Triton X-100-soluble to the Triton X-100-insoluble fractions. These morphological changes are associated with a decrease in transendothelial electrical membrane resistance and an increase in [14C]inulin permeability. MCP-1 did not induce these events in brain endothelial cells prepared from mice genotype CCR2–/–. The Rho kinase inhibitor Y27632 and inhibition of Rho (C3 exoenzyme, and dominant negative mutant of Rho, RhoT19N) prevented MCP-1-induced stress fiber assembly, reorganization of tight junction proteins and alterations in endothelial permeability. In all, this suggests that a small GTPase Rho and Rho kinase have a pivotal role in MCP-1-induced junction disarrangement. These data are the first to strongly suggest that MCP-1, via CCR2 present on brain endothelial cells, contributes to increased brain endothelial permeability.

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.

Acute inflammatory reaction following experimental intracerebral hemorrhage in rat.

Previous studies on intracerebral hemorrhage (ICH) indicate that brain edema increases progressively in the first 24 h and remains elevated for several days. The cause of secondary brain injury and edema formation is uncertain. We hypothesized that inflammatory mediators released from the blood after cerebral hemorrhage might cause secondary brain injury and edema formation. This study investigates if, when and where inflammation occurs after ICH in rat. Immunocytochemistry for polymorphonuclear leukocyte marker (myeloperoxidase, MPO), microglia marker (OX42) and intracellular adhesion molecule-1 (ICAM-1) was performed in control, and 1, 3, 7 and 10 days after the injection of 100 microliter autologous blood in the right basal ganglia. Double labeling immunohistochemistry was used to identify ICAM-1 positive cells. The results show that an inflammatory response occurred in and around the blood clot after ICH, characterized by the infiltration of neutrophils and macrophages as well as activation of microglia. ICAM-1 immunoreactivity was observed in blood vessels adjacent to the clot, as well as in activated microglia and neurons in the ipsilateral hemisphere. The present study demonstrates there is an inflammatory response in the brain after ICH. Infiltrating leukocytes and activated microglia may release cytotoxic mediators contributing to secondary brain injury.

Absence of the Chemokine Receptor CCR2 Protects Against Cerebral Ischemia/Reperfusion Injury in Mice

Background and Purpose— The chemokine, monocyte chemoattractant protein-1 (CCL2), is a major factor driving leukocyte infiltration into the brain parenchyma in a variety of neuropathologic conditions associated with inflammation, including stroke. In addition, recent studies indicate that CCL2 and its receptor (CCR2) could have an important role in regulating blood-brain barrier (BBB) permeability. This study evaluated the role of the CCL2/CCR2 axis in regulating postischemic inflammation, BBB breakdown, and vasogenic edema formation. Methods— CCR2−/− and CCR2+/+ mice were subjected to focal transient cerebral ischemia. BBB permeability and brain edema formation were observed at days 1 and 5 of reperfusion by evaluating the product surface area for fluorescein isothiocyanate–albumin and measuring water and electrolyte contents. Immunohistochemistry was used to assess leukocyte infiltration. cDNA gene and protein arrays for inflammatory cytokines were used to assess inflammatory profiles in CCR2+/+ and CCR2−/− mice. Results— CCR2−/− mice had reduced infarct sizes and significantly reduced BBB permeability and brain edema formation in the affected ischemic hemisphere compared with CCR2+/+ mice. This reduction in injury was closely associated with reduced infiltration of not only monocytes but also neutrophils (7- and 4-fold decreases, respectively). In addition, CCR2−/− mice had reduced expression/production of inflammatory cytokines during reperfusion. Conclusions— These data suggest that inhibiting the CCL2/CCR2 axis affects brain reperfusion outcome by reducing brain edema, leukocyte infiltration, and inflammatory mediator expression.

Brain endothelial cell-cell junctions: how to "open" the blood brain barrier.

The blood-brain barrier (BBB) is a highly specialized structural and biochemical barrier that regulates the entry of blood-borne molecules into brain, and preserves ionic homeostasis within the brain microenvironment. BBB properties are primarily determined by junctional complexes between the cerebral endothelial cells. These complexes are comprised of tight and adherens junctions. Such restrictive angioarchitecture at the BBB reduces paracellular diffusion, while minimal vesicle transport activity in brain endothelial cells limits transcellular transport. Under normal conditions, this largely prevents the extravasation of large and small solutes (unless specific transporters are present) and prevents migration of any type of blood-borne cell. However, this is changed in many pathological conditions. There, BBB disruption (“opening”) can lead to increased paracellular permeability, allowing entry of leukocytes into brain tissue, but also contributing to edema formation. In parallel, there are changes in the endothelial pinocytotic vesicular system resulting in the uptake and transfer of fluid and macromolecules into brain parenchyma. This review highlights the route and possible factors involved in BBB disruption in a variety of neuropathological disorders (e.g. CNS inflammation, Alzheimer’s disease, Parkinson’s disease, epilepsy). It also summarizes proposed signal transduction pathways that may be involved in BBB “opening”.