Anthony J. Williams

Quantitative Real-Time RT—PCR Analysis of Inflammatory Gene Expression Associated with Ischemia—Reperfusion Brain Injury

Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines, some of which are regulated by the nuclear transcription factor NF-κB. In this study the authors examined mRNA expression levels for several important genes associated with inflammation at five time points (3, 6, 12, 24, and 72 hours) after transient middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. A sensitive and quantitative technique (TaqMan real-time QRT-PCR) was used to simultaneously measure mRNA levels for key cell adhesion molecules and inflammatory cytokines. Gene expression increased significantly in the injured hemisphere for interleukin (IL)-1β (12-fold increase at 24 hours), IL-6 (25-fold increase at 6 hours) and ICAM-1 (4-fold increase at 24 hours), and the in-terhemispheric differences for these genes were significant for every time point examined (P < 0.05 for all values). Tumor necrosis factor-α mRNA was upregulated in the injured versus uninjured hemisphere from 3 to 24 hours (5-fold increase at 6 hours), while E-selectin showed a significant increase in mRNA levels from 6 to 24 hours after MCAO (10-fold increase at 6 hours) (P < 0.05 for all values). VCAM-1 mRNA levels did not respond differentially to injury at any time point between the two brain hemispheres. At all time points examined, activated NF-κB immunoreactivity was observed in cells throughout the infarct-damaged tissue. These results are consistent with the proinflammatory properties of the induced molecules, which are involved in the initiation of the inflammatory cascade, and may thus contribute to secondary cellular responses that lead to further brain damage.

Proteasome Inhibitor PS519 Reduces Infarction and Attenuates Leukocyte Infiltration in a Rat Model of Focal Cerebral Ischemia

BACKGROUND AND PURPOSE Reperfusion brain injury after cerebral ischemia is associated with a developing inflammatory response at the site of infarction. Proteasome inhibitors block nuclear factor-kappaB activation and provide anti-inflammatory effects in several animal models of peripheral inflammation. We tested the novel proteasome inhibitor PS519 in a rat model of transient focal ischemia to establish its pharmacodynamics as a neuroprotection treatment and related effects on leukocyte infiltration. METHODS Rats were subjected to 2 hours of focal cerebral ischemia by means of the filament method of middle cerebral artery occlusion (MCAo). After either 22 or 70 hours of reperfusion, infarct size was measured and neurological function, electroencephalographic (EEG) activity, and/or neutrophil and macrophage infiltration was quantified. PS519 was administered in a single intravenous bolus at 2 hours after MCAo. In addition, the therapeutic window for PS519 was estimated by delaying treatment for 4 or 6 hours after MCAo. RESULTS Dose-response analysis of infarct volume at 24 hours revealed that PS519 neuroprotection approached 60%, and clinical evaluations showed significant improvements in neurological function and EEG activity. Neutrophil infiltration at 24 hours was also significantly decreased in cortical and striatal infarcted tissue of PS519-treated rats. Delaying the PS519 treatment up to 4 hours continued to result in significant neuroprotection. In the 72-hour injury model, infarction was reduced 40% by PS519, and significant improvements in neurological function and EEG recovery were again measured. Considerable reductions in both neutrophil and macrophage infiltration were evident. CONCLUSIONS PS519 mitigates infarction and improves neurological recovery in brain-injured rats, an effect in part caused by a reduction in the leukocyte inflammatory response.

Microarray analysis of acute and delayed gene expression profile in rats after focal ischemic brain injury and reperfusion.

Temporal changes in gene expression were measured using DNA microarrays after 30‐min or 2‐hr transient middle cerebral artery occlusion (MCAo) in rats. Total RNA was extracted from the injured hemisphere at 30 min, 4 hr, 8 hr, 24 hr, 3 days, and 7 days after MCAo for GeneChip analysis using Affymetrix U34 Rat Neurobiology arrays (1,322 functional genes). In total, 267 genes were expressed differentially: 166 genes were upregulated, 94 genes were downregulated, and 7 genes were biphasically up‐ and downregulated. Among all differentially expressed genes, 88 were newly identified as associated with ischemic brain injury. Most affected genes were distributed among 12 functional categories. Immediate early genes, transcription factors, and heat shock proteins were upregulated as early as 30 min after MCAo, followed by the upregulation of inflammation, apoptosis, cytoskeletal, and metabolism genes, which peaked within 4–24 hr of injury. Neurotrophic growth factors exhibited a sustained upregulation beginning 24 hr after MCAo and persisting through 7 days post‐injury. Three classes of genes were downregulated with distinct temporal patterns: ion channel genes and neurotransmitter receptor genes were downregulated between 8–24 hr after injury, whereas synaptic proteins genes were downregulated between 3–7 days after MCAo. Downregulation of synaptic protein gene expression after ischemic injury is of particular interest because of its conspicuously delayed pattern as a functional group, which has not been reported previously and may play a role in post‐injury recovery.

Occurrence of nonconvulsive seizures, periodic epileptiform discharges, and intermittent rhythmic delta activity in rat focal ischemia.

A significant proportion of neurologic patients suffer electroencephalographic (EEG) seizures in the acute phase following traumatic or ischemic brain injury, including many without overt behavioral manifestations. Although such nonconvulsive seizures may exacerbate neuropathological processes, they have received limited attention clinically and experimentally. Here we characterize seizure episodes following focal cerebral ischemia in the rat as a model for brain injury-induced seizures. Cortical EEG activity was recorded continuously from both hemispheres up to 72 h following middle cerebral artery occlusion (MCAo). Seizure discharges appeared in EEG recordings within 1 h of MCAo in 13/16 (81%) animals and consisted predominantly of generalized 1-3 Hz rhythmic spiking. During seizures animals engaged in quiet awake or normal motor behaviors, but exhibited no motor convulsant activity. Animals had a mean of 10.6 seizure episodes within 2 h, with a mean duration of 60 s per episode. On average, seizures ceased at 1 h 59 min post-MCAo in permanently occluded animals and did not occur following reperfusion at 2 h in transiently occluded animals. In addition to seizures, periodic lateralized epileptiform discharges (PLEDs) appeared over penumbral regions in the injured hemisphere while intermittent rhythmic delta activity (IRDA) recurred in the contralateral hemisphere with frontoparietal dominance. PLEDs and IRDA persisted up to 72 h in permanent MCAo animals, and early onset of the former was predictive of prolonged seizure activity. The presentation of these EEG waveforms, each with characteristic features replicating those in clinical neurologic populations, validates rat MCAo for study of acutely induced brain seizures and other neurophysiological aspects of brain injury.

Acute and delayed neuroinflammatory response following experimental penetrating ballistic brain injury in the rat

BackgroundNeuroinflammation following acute brain trauma is considered to play a prominent role in both the pathological and reconstructive response of the brain to injury. Here we characterize and contrast both an acute and delayed phase of inflammation following experimental penetrating ballistic brain injury (PBBI) in rats out to 7 days post-injury.MethodsQuantitative real time PCR (QRT-PCR) was used to evaluate changes in inflammatory gene expression from the brain tissue of rats exposed to a unilateral frontal PBBI. Brain histopathology was assessed using hematoxylin and eosin (H&E), silver staining, and immunoreactivity for astrocytes (GFAP), microglia (OX-18) and the inflammatory proteins IL-1β and ICAM-1.ResultsTime course analysis of gene expression levels using QRT-PCR indicated a peak increase during the acute phase of the injury between 3–6 h for the cytokines TNF-α (8–11 fold), IL-1β (11–13 fold), and IL-6 (40–74 fold) as well as the cellular adhesion molecules VCAM (2–3 fold), ICAM-1 (7–15 fold), and E-selectin (11–13 fold). Consistent with the upregulation of pro-inflammatory genes, peripheral blood cell infiltration was a prominent post-injury event with peak levels of infiltrating neutrophils (24 h) and macrophages (72 h) observed throughout the core lesion. In regions of the forebrain immediately surrounding the lesion, strong immunoreactivity for activated astrocytes (GFAP) was observed as early as 6 h post-injury followed by prominent microglial reactivity (OX-18) at 72 h and resolution of both cell types in cortical brain regions by day 7. Delayed thalamic inflammation (remote from the primary lesion) was also observed as indicated by both microglial and astrocyte reactivity (72 h to 7 days) concomitant with the presence of fiber degeneration (silver staining).ConclusionIn summary, PBBI induces both an acute and delayed neuroinflammatory response occurring in distinct brain regions, which may provide useful diagnostic information for the treatment of this type of brain injury.

Selective mGluR5 receptor antagonist or agonist provides neuroprotection in a rat model of focal cerebral ischemia

Activation of group I metabotropic glutamate receptors (mGluR) has been implicated in the pathophysiology of acute central nervous system injury. However, the relative roles of the two group I subtypes, mGluR1 or mGluR5, in such injury has not been well examined. We compared the effects of treatment with the newly developed, selective mGluR5 antagonist 2-methyl-6-phenylethynylpyridine (MPEP) and the selective mGluR5 agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) in a rat intraluminal filament model of temporary middle cerebral artery occlusion (MCAo). Rats were administered MPEP or CHPG i.c.v. beginning 15 or 135 min after induction of ischemia for 2 h. Infarct size was measured after either 22 or 70 h of reperfusion, and neurological function was quantified at 2, 24, 48 and 72 h. Treatment with MPEP or CHPG at 15 min reduced 24 h infarct volume by 61 and 44%, respectively. The neuroprotective effects were dose dependent. Delaying MPEP treatment until 135 min eliminated the neuroprotective effects. In other studies, using early MPEP treatment (15 min) at optimal doses, infarct volume was reduced by 44% at 72 h and this was correlated with significant neurological recovery. These data suggest that both MPEP and CHPG are neuroprotective when administered after focal cerebral ischemia. In separate, recent studies we found that although MPEP does act as an mGluR5 antagonist and blocks agonist induced phosphoinositide hydrolysis, it also serves as a non-competitive NMDA antagonist; in contrast, other results indicate that CHPG mediated neuroprotection may reflect anti-apoptotic activity. Therefore, both types of compounds may prove to have therapeutic potential for the treatment of stroke.

Neuroprotection with the proteasome inhibitor MLN519 in focal ischemic brain injury: Relation to nuclear factor κB (NF-κB), inflammatory gene expression, and leukocyte infiltration

The ubiquitin proteasome system (UPS) is a major cellular protein degradation pathway that involves the modulation of key proteins controlling inflammation, cell cycle regulation and gene expression. Modulation of the UPS with proteasome inhibitors has indicated efficacy in the treatment of several disease states including cancer and neuro-inflammatory disorders. In particular, a series of recent reports have evaluated the pre-clinical efficacy of the proteasome inhibitor MLN519 for the treatment of focal ischemic/reperfusion brain injury in rats. Evidence from these studies indicate that the neuroprotection provided by MLN519 is related to an anti-inflammatory effect linked to the modulation of nuclear factor kappaB (NF-kappaB) activity, attenuation of cytokine (TNF-alpha, IL-1beta, and IL-6) and cellular adhesion molecule (ICAM-1 and E-selectin) expression, and reduction of neutrophil and macrophage infiltration into the injured rat brain. It is the aim of this paper to review the experimental neuroprotection data reported using MLN519 with a focus on the molecular and cellular mechanisms of anti-inflammatory action.

Delayed Treatment of Ischemia/Reperfusion Brain Injury Extended Therapeutic Window with the Proteosome Inhibitor MLN519

Background and Purpose— Clinical development of novel neuroprotection therapies for the treatment of brain injury has been unsuccessful. One critical limitation is the lack of a viable therapeutic treatment window (TW). In this study, we evaluated the neuroprotection TW for the proteosome inhibitor MLN519 after ischemia/reperfusion brain injury in rats as related to its antiinflammatory mechanism. Methods— Male Sprague-Dawley rats were subjected to 2 hours of middle cerebral artery occlusion (MCAo), followed by 70 hours of reperfusion and recovery. MLN519 was administered after injury (starting 6 to 12 hours after MCAo) to evaluate the full TW. Brain infarction, neuronal degeneration, neurological recovery, leukocyte infiltration, and inflammatory gene mRNA levels were assessed. Results— Core infarct volume in vehicle-treated rats (216 ± 25 mm3) was reduced with delayed MLN519 treatments of 6, 8, or 10 hours after injury (45 ± 13, 86 ± 28, and 150 ± 27 mm3, respectively, P < 0.05) and was associated with reductions in neuronal and axonal degeneration. MLN519-treated rats had reduced brain mRNA levels of TNF-&agr;(46%, P < 0.05), ICAM-1 (58%, P < 0.05), IL-6 (58%, P < 0.05), and E-selectin (72%, P < 0.05) at 24 hours after injury. Furthermore, MLN519 treatment reduced leukocyte infiltration by 32% to 80% (P < 0.05) in ischemic brain regions. Conclusions— Neuroprotection treatment with MLN519 provides an extended TW of up to 10 hours after ischemia/reperfusion brain injury, in part by attenuating the inflammatory response. As such, the delayed onset of brain inflammation after an ischemic injury offers a prime target for extending the neuroprotective TW with compounds such as MLN519, used either alone or possibly as an adjunctive therapy with thrombolytic agents.

Delayed Treatment with MLN519 Reduces Infarction and Associated Neurologic Deficit Caused by Focal Ischemic Brain Injury in Rats via Antiinflammatory Mechanisms Involving Nuclear Factor-κB Activation, Gliosis, and Leukocyte Infiltration

Secondary brain injury due to ischemia includes the infiltration of leukocytes into the brain parenchyma mediated by activation of nuclear factor-κB (NF-κB), which is activated by proteasome degradation. Neuroprotection with the proteasome inhibitor MLN519 has previously been reported to decrease ischemic brain injury in rats. The authors used higher doses of MLN519 to evaluate the neuroprotection therapeutic window after 24 hours of brain injury in rats as correlated to proteasome levels, activated NF-κB immunoreactivity, and leukocyte infiltration. Male Sprague-Dawley rats were subjected to 2-hour middle cerebral artery occlusion (MCAO) and recovery. MLN519 or vehicle was administered after injury with a single injection given in delayed increments of 2 hours (i.e., 4, 6, or 8 hours after MCAO). Treatment with MLN519 up to 6 hours after MCAO (4 hours after reperfusion) effectively reduced neuronal and astrocytic degeneration, decreased cortical infarct volume, and increased neurologic recovery. These effects were related to >80% reductions in blood proteasome levels, reduced neutrophil infiltration, and a decrease in activated NF-κB immunoreactivity. This improved neuroprotection profile and antiinflammatory effect of MLN519 provides an exciting avenue for potential treatment of focal ischemic brain injury in humans.

Central neuro-inflammatory gene response following soman exposure in the rat.

Effective treatments to improve survivability following exposure to the nerve agent soman have been established and are currently available. Unfortunately, electrographic brain seizures, neuroinflammation and brain cell death are still a potential problem even with treatment. In the present study we have characterized the time course of the central neuro-inflammatory gene response using quantitative real time-PCR (TaqMan). Male Sprague-Dawley rats were pre-treated with HI-6 (1-2-hydroxy-iminomethyl-1-pyridino-3-(4-carbamoyl-1-pyridino-2-oxapropane dichloride); 125 mg/kg, i.p.) and exposed 30 min later to 1.6 x LD(50) of soman (pinacolyl methyl-phosphonofluoridate, 180 microg/kg, s.c.) followed at 1 min by atropine methyl nitrate (4 mg/kg, i.m.). Initially, a significant and dramatic upregulation of tumor necrosis factor-alpha and vascular cell adhesion molecule-1 mRNA levels was measured 2 h post-exposure followed at 6 h by upregulation of interleukin-1beta, interleukin-6, E-selectin, and intercellular adhesion molecule-1 with eventual resolution by 24-48 h. In conclusion, an acute and transient upregulation of the inflammatory gene response is activated following soman exposure that may be involved in the soman-induced brain injury process.