Toll-like receptor 4 contributes to poor outcome after intracerebral hemorrhage.

OBJECTIVE: Intracerebral hemorrhage (ICH) is a devastating stroke subtype in which perihematomal inflammation contributes to neuronal injury and functional disability. Histologically, the region becomes infiltrated with neutrophils and activated microglia followed by neuronal loss, but little is known about the immune signals that coordinate these events. This study aimed to determine the role of Toll-like receptor 4 (TLR4) in the innate immune response after ICH and its impact on neurobehavioral outcome. METHODS: Transgenic mice incapable of TLR4 signaling and wild-type controls were subjected to striatal blood injection to model ICH. The perihematomal inflammatory response was then quantified by immunohistochemistry, whole brain flow cytometry, and polymerase chain reaction. The critical location of TLR4 signaling was determined by blood transfer experiments between genotypes. Functional outcomes were quantified in all cohorts using the cylinder and open field tests. RESULTS: TLR4-deficient mice had markedly decreased perihematomal inflammation, associated with reduced recruitment of neutrophils and monocytes, fewer microglia, and improved functional outcome by day 3 after ICH. Moreover, blood transfer experiments revealed that TLR4 on leukocytes or platelets within the hemorrhage contributes to perihematomal leukocyte infiltration and the neurological deficit. INTERPRETATION: Together, these data identify a critical role for TLR4 signaling in perihematomal inflammation and injury and indicate this pathway may be a target for therapeutic intervention.

Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.

In vitro-transcribed mRNAs encoding physiologically important proteins have considerable potential for therapeutic applications. However, in its present form, mRNA is unfeasible for clinical use because of its labile and immunogenic nature. Here, we investigated whether incorporation of naturally modified nucleotides into transcripts would confer enhanced biological properties to mRNA. We found that mRNAs containing pseudouridines have a higher translational capacity than unmodified mRNAs when tested in mammalian cells and lysates or administered intravenously into mice at 0.015-0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after the injection, where both products were at significantly higher levels when pseudouridine-containing mRNA was administered. Even at higher doses, only the unmodified mRNA was immunogenic, inducing high serum levels of interferon-alpha (IFN-alpha). These findings indicate that nucleoside modification is an effective approach to enhance stability and translational capacity of mRNA while diminishing its immunogenicity in vivo. Improved properties conferred by pseudouridine make such mRNA a promising tool for both gene replacement and vaccination.

Cerebral preconditioning using cortical application of hypertonic salt solutions: upregulation of mRNAs encoding inhibitors of inflammation.

Previous studies have demonstrated that local application of hypertonic KCl or NaCl to the cerebral cortex induces tolerance to a subsequent episode of ischemia. The objective of the present study was to determine whether application of these salts increases the levels of mRNAs encoding inhibitors of inflammation. Hypertonic KCl or NaCl was applied for 2 h to the frontal cortex of Sprague-Dawley rats. After recovery periods up to 24 h, levels of selected mRNAs were measured in samples from frontal and parietal cortex using Northern blots. Application of hypertonic KCl caused a rapid and widespread increase in the levels of mRNA coding for tumor necrosis factor (TNF), tristetraprolin (TTP), suppressor of cytokine signaling-3 (SOCS3), and brain-derived neurotrophic factor (BDNF), and a 24-h delayed induction of ciliary neurotrophic factor (CNTF) mRNA. Application of hypertonic NaCl caused alterations in mRNA levels that were restricted to the frontal cortex. In this region, application of NaCl rapidly increased levels of mRNA encoding TNF, TTP, and SOCS3, but not BDNF, and caused a delayed induction of CNTF mRNA. These results raise the possibility that upregulation of inhibitors of inflammation after preconditioning may contribute to the induction of tolerance to ischemia.

Inhibition of toll-like receptor and cytokine signaling--a unifying theme in ischemic tolerance.

Cerebral ischemia triggers acute inflammation, which exacerbates primary brain damage. Activation of the innate immune system is an important component of this inflammatory response. Inflammation occurs through the action of proinflammatory cytokines, such as TNF, IL-1 beta and IL-6, that alter blood flow and increase vascular permeability, thus leading to secondary ischemia and accumulation of immune cells in the brain. Production of these cytokines is initiated by signaling through Toll-like receptors (TLRs) that recognize host-derived molecules released from injured tissues and cells. Recently, great strides have been made in understanding the regulation of the innate immune system, particularly the signaling mechanisms of TLRs. Negative feedback inhibitors of TLRs and inflammatory cytokines have now been identified and characterized. It is also evident that lipid rafts exist in membranes and play a role in receptor-mediated inflammatory signaling events. In the present review, using this newly available large body of knowledge, we take a fresh look at studies of ischemic tolerance. Based on this analysis, we recognize a striking similarity between ischemic tolerance and endotoxin tolerance, an immune suppressive state characterized by hyporesponsiveness to lipopolysaccharide (LPS). In view of this analogy, and considering recent discoveries related to molecular mechanisms of endotoxin tolerance, we postulate that inhibition of TLR and proinflammatory cytokine signaling contributes critically to ischemic tolerance in the brain and other organs. Ischemic tolerance is a protective mechanism induced by a variety of preconditioning stimuli. Tolerance can be established with two temporal profiles: (i) a rapid form in which the trigger induces tolerance to ischemia within minutes and (ii) a delayed form in which development of protection takes several hours or days and requires de-novo protein synthesis. The rapid form of tolerance is achieved by direct interference with membrane fluidity, causing disruption of lipid rafts leading to inhibition of TLR/cytokine signaling pathways. In the delayed form of tolerance, the preconditioning stimulus first triggers the TLR/cytokine inflammatory pathways, leading not only to inflammation but also to simultaneous upregulation of feedback inhibitors of inflammation. These inhibitors, which include signaling inhibitors, decoy receptors, and anti-inflammatory cytokines, reduce the inflammatory response to a subsequent episode of ischemia. This novel interpretation of the molecular mechanism of ischemic tolerance highlights new avenues for future investigation into the prevention and treatment of stroke and related diseases.

Cortical spreading depression causes a long-lasting decrease in cerebral blood flow and induces tolerance to permanent focal ischemia in rat brain.

Cortical spreading depression (CSD) has previously been shown to induce tolerance to a subsequent episode of transient cerebral ischemia. The objective of the present study was to determine whether CSD also induces tolerance to permanent focal ischemia and, if so, whether tolerance may be mediated by alterations in cerebral blood flow (CBF). Sprague-Dawley rats were preconditioned by applying potassium chloride to one hemisphere for 2 hours, evoking 19 +/- 5 episodes of CSD (mean +/- SD, n = 19). Three days later, the middle cerebral artery (MCA) was permanently occluded using an intraluminal suture. In a subset of animals, laser Doppler blood flow (LDF) was monitored over the parietal cortex before and during the first 2 hours of MCA occlusion. Preconditioning with CSD reduced the hemispheric volume of infarction from 248 +/- 115 mm3 (n = 18) in sham-conditioned animals to 161 +/- 81 mm3 (n = 19, P< 0.02). Similarly, CSD reduced the neocortical volume of infarction from 126 +/- 82 mm3 to 60 +/- 61 mm3 (P < 0.01). Moreover, preconditioning with CSD significantly improved LDF during MCA occlusion from 21% +/- 7% (n = 9) of preischemic baseline in sham-conditioned animals to 29% +/- 9% (n = 7, P< 0.02). Preconditioning with CSD therefore preserved relative levels of CBF during focal ischemia and reduced the extent of infarction resulting from permanent MCA occlusion. To determine whether CSD may have altered preischemic baseline CBF, [14 C]iodoantipyrine was used in additional animals to measure CBF 3 days after CSD conditioning or sham conditioning. CSD, but not sham conditioning, significantly reduced baseline CBF in the ipsilateral neocortex to values 67% to 75% of those in the contralateral cortex. Therefore, CSD causes a long-lasting decrease in baseline CBF that is most likely related to a reduction in metabolic rate. A reduction in the rate of metabolism may contribute to the induction of tolerance to ischemia after preconditioning with CSD.

Induction of tolerance to focal ischemia in rat brain: dissociation between cortical lesioning and spreading depression.

Cortical application of KCl has previously been shown to induce tolerance to a subsequent episode of cerebral ischemia. KCl triggers recurrent episodes of cortical spreading depression and produces a small lesion at the cortical application site. To determine whether a cortical lesion alone is sufficient to induce tolerance to ischemia, the authors used 5-mol/L NaCl to precondition rat brain 3 days before permanent occlusion of the middle cerebral artery. NaCl produced a small lesion at the application site without evoking cortical spreading depression. Preconditioning with 5-mol/L NaCl significantly attenuated the decrease in CBF after middle cerebral artery occlusion and reduced the volume of cortical infarction by 35%. The results show that a small cortical lesion, by itself, is sufficient to induce tolerance to ischemia.

Structural and functional damage sustained by mitochondria after traumatic brain injury in the rat: evidence for differentially sensitive populations in the cortex and hippocampus.

The cellular and molecular pathways initiated by traumatic brain injury (TBI) may compromise the function and structural integrity of mitochondria, thereby contributing to cerebral metabolic dysfunction and cell death. The extent to which TBI affects regional mitochondrial populations with respect to structure, function, and swelling was assessed 3 hours and 24 hours after lateral fluid-percussion brain injury in the rat. Significantly less mitochondrial protein was isolated from the injured compared with uninjured parietotemporal cortex, whereas comparable yields were obtained from the hippocampus. After injury, cortical and hippocampal tissue ATP concentrations declined significantly to 60% and 40% of control, respectively, in the absence of respiratory deficits in isolated mitochondria. Mitochondria with ultrastructural morphologic damage comprised a significantly greater percent of the population isolated from injured than uninjured brain. As determined by photon correlation spectroscopy, the mean mitochondrial radius decreased significantly in injured cortical populations (361 +/- 40 nm at 24 hours) and increased significantly in injured hippocampal populations (442 +/- 36 at 3 hours) compared with uninjured populations (Ctx: 418 +/- 44; Hipp: 393 +/- 24). Calcium-induced deenergized swelling rates of isolated mitochondrial populations were significantly slower in injured compared with uninjured samples, suggesting that injury alters the kinetics of mitochondrial permeability transition (MPT) pore activation. Cyclosporin A (CsA)-insensitive swelling was reduced in the cortex, and CsA-sensitive and CsA-insensitive swelling both were reduced in the hippocampus, demonstrating that regulated MPT pores remain in mitochondria isolated from injured brain. A proposed mitochondrial population model synthesizes these data and suggests that cortical mitochondria may be depleted after TBI, with a physically smaller, MPT-regulated population remaining. Hippocampal mitochondria may sustain damage associated with ballooned membranes and reduced MPT pore calcium sensitivity. The heterogeneous mitochondrial response to TBI may underlie posttraumatic metabolic dysfunction and contribute to the pathophysiology of TBI.

Traumatic brain injury elevates glycogen and induces tolerance to ischemia in rat brain.

Previous studies have demonstrated that traumatic brain injury (TBI) increases the vulnerability of the brain to an acute episode of hypoxia-ischemia. The objective of the present study was to determine whether TBI alters the vulnerability of the brain to a delayed episode of ischemia and, if so, to identify contributing mechanisms. Sprague-Dawley rats were subjected to lateral fluid-percussion (FP) brain injury (n = 14) of moderate severity (2.3-2.5 atm), or sham-injury (n = 12). After recovery for 24 h, all animals underwent an 8-min episode of forebrain ischemia, followed by survival for 6 days. Ischemic damage in the hippocampus and cerebral cortex of the FP-injured hemisphere was compared to that in the contralateral hemisphere and to that in sham-injured animals. Remarkably, the number of surviving CA(1) neurons in the middle and lateral segments of the hippocampus in the FP-injured hemisphere was significantly greater than that in the contralateral hemisphere and sham-injured animals (p < 0.05). Likewise, in the cerebral cortex the number of damaged neurons tended to be lower in the FP-injured hemisphere than in the contralateral hemisphere. These results suggest that TBI decreased the vulnerability of the brain to a delayed episode of ischemia. To determine whether TBI triggers protective metabolic alterations, glycogen levels were measured in cerebral cortex and hippocampus in additional animals 24 h after FP-injury (n = 13) or sham-injury (n = 7). Cortical glycogen levels in the ipsilateral hemisphere increased to 12.9 +/- 6.4 mmol/kg (mean +/- SD), compared to 6.4 +/- 1.8 mmol/kg in the opposite hemisphere and 5.7 +/- 1.3 mmol/kg in sham-injured animals (p < 0.001). Similarly, in the hippocampus glycogen levels in the FP-injured hemisphere increased to 13.4 +/- 4.9 mmol/kg, compared to 8.1 +/- 2.4 mmol/kg in the contralateral hemisphere (p < 0.004) and 6.2 +/- 1.5 mmol/kg in sham-injured animals (p < 0.001). These results demonstrate that TBI triggers a marked accumulation of glycogen that may protect the brain during ischemia by serving as an endogenous source of metabolic energy.

Autologous blood injection to model spontaneous intracerebral hemorrhage in mice.

Investigation of the pathophysiology of injury after intracerebral hemorrhage (ICH) requires a reproducible animal model. While ICH accounts for 10-15% of all strokes, there remains no specific effective therapy. The autologous blood injection model in mice involves the stereotaxic injection of arterial blood into the basal ganglia mimicking a spontaneous hypertensive hemorrhage in man. The response to hemorrhage can then be studied in vivo and the neurobehavioral deficits quantified, allowing for description of the ensuing pathology and the testing of potential therapeutic agents. The procedure described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.