Rossana Berti

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.

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.

High Frequency of Human Herpesvirus 6 DNA in Multiple Sclerosis Plaques Isolated by Laser Microdissection

The frequency of human herpesvirus 6 (HHV-6) DNA was assessed in autopsy material from multiple sclerosis (MS) plaques and normal-appearing white matter (NAWM) from brains of persons with MS, healthy brains, and brains of persons with other neurologic diseases. Specific areas from formalin-fixed, paraffin-embedded brain tissue samples were isolated by laser microscope. DNA was extracted from laser microdissected brain material, and HHV-6 genomic sequences were amplified by nested polymerase chain reaction. We analyzed 44 NAWM samples and 64 MS plaques from 13 patients with MS, 46 samples from 13 patients with non-MS neurologic disorders, and 41 samples from 12 healthy control brains. Of the 44 NAWM samples, 7 (15.9%) were positive for HHV-6 DNA sequences, versus 37 (57.8%) of 64 MS plaques (P<.0005). HHV-6 DNA was detected in 10 (21.7%) of 46 samples from patients with non-MS neurologic disorders and in 11 (26.8%) of 41 samples from patients without known neurologic disease. Although the frequency of HHV-6 DNA did not differ significantly by sample type, HHV-6 DNA was significantly more common in MS plaques, suggesting that HHV-6 may play a role in MS pathogenesis.

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.

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.

Effect of the proteasome inhibitor MLN519 on the expression of inflammatory molecules following middle cerebral artery occlusion and reperfusion in the rat

Anti-inflammatory treatment with the proteasome inhibitor MLN519 has been previously reported to be neuroprotective against ischemic brain injury in rats. These effects have been related to inhibition of the transcription factor NF-κB, which is activated through ubiquitin-proteasomal degradation. The aim of this study was to evaluate the effects of MLN519 to alter the expression of several inflammatory genes under the control of NF-κB. Male Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAo) followed by vehicle or MLN519 (1.0 mg/kg, i.v.) treatment immediately after reperfusion of blood to the brain at 2h. Gene expression was evaluated 3–72 h post-MCAo. The most striking effects of intravenous treatment with MLN519 were associated with reductions in ICAM-1 expression at 3 h followed by reductions in E-selectin (12–72 h). Less dramatic reductions were observed in IL-1β (3–24h) and TNF-α (24 h) with no apparent effects on IL-6 and VCAM-1 mRNA levels. Immunohistochemical analysis revealed that the genes most dramatically affected by MLN519 had highest expression in endothelial cells and leukocytes (E-selectin, ICAM-1), indicating that these cell types may be the primary targets of intravenously delivered MLN519 treatment.

Differential Pattern of Expression of Voltage-gated Sodium Channel Genes Following Ischemic Brain Injury in Rats †

This study investigated the effects of brain ischemia on sodium channel gene (NaCh) expression in rats. Using quantitative RT-PCR, our findings demonstrated the expression ratio of NaCh genes in normal rat brain to be Nav1.1>Nav1.8>Nav1.3>Nav1.7 (rBI>PN3>rBIII>PN1). In contrast, brain injury caused by middle cerebral artery occlusion (MCAo) for 2h followed by reperfusion significantly down-regulated Nav1.3 and Nav1.7 genes in both injured and contralateral hemispheres; whereas the Nav1.8 gene was down regulated in only the injured hemisphere (though only acutely at 2 or 2–6 h post-MCAo). However, the time-course of NaCh gene expression revealed a significant down-regulation of Nav1.1 only in the ischemic hemisphere beginning 6h post-MCAo and measured out to 48h post-MCAo. In a separate preliminary study Nav1.2 (rBII) gene was found be expressed at levels greater than that of Nav1.1 in normal rats and was significantly down regulated at 24h post-MCAo. Our findings document, for the first time, quantitative and relative changes in the expression of various NaCh genes following ischemic brain injury and suggest that the Nav1.1 sodium channel gene may play a key role in ischemic injury/recovery.

The sodium channel blocker RS100642 reverses down-regulation of the sodium channel α-subunit Nav 1.1 expression caused by transient ischemic brain injury in rats

In this study we evaluated the expression of five sodium channel (NaCh) α-subunit genes after transient middle cerebral artery occlusion (MCAo) in the rat and the effects of treatment with the NaCh blocker and experimental neuroprotective agent RS100642 as compared to the prototype NaCh blocker mexiletine. The expression of Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.7, Nav 1.8 and the housekeeping gene β-actin were studied in vehicle or drug-treated rats at 6, 24 and 48 h post-MCAo using real-time quantitative RT-PCR. RS100642 (1 mg/kg), mexiletine (10 mg/kg), or vehicle (1 ml/kg) was injected (i.v.) at 30 min, 2, 4, and 6 h post-injury. Following MCAo only the Nav 1.1. and Nav 1.2 genes were significantly down-regulated in the ipsilateral hemisphere of the injured brains. RS100642 treatment significantly reversed the down-regulation of Nav 1.1 (but not Nav 1.2) at 24–48 h post-injury. Mexiletine treatment, on the other hand, had no significant effect on the down-regulation of either gene. These findings demonstrate that treatment with a neuroprotective dose of RS100642 significantly reverses the down-regulation of Nav 1.1 caused by ischemic brain injury and suggests that RS100642 selectively targets the Nav 1.1 α-subunit of the NaCh. Furthermore, our findings strengthen the hypothesis that ischemic injury may produce selective depletion of voltage-gated NaChs, and suggest that the Nav 1.1 NaCh α-subunit may play a key role in the neuronal injury/recovery process.

Down-regulation of sodium channel Nav1.1 expression by veratridine and its reversal by a novel sodium channel blocker, RS100642, in primary neuronal cultures

This study investigated the effects of veratridine-induced neuronal toxicity on sodium channel gene (NaCh) expression in primary forebrain cultures enriched in neurons, and its reversal by a novel sodium channel blocker, RS100642. Using quantitative RTPCR, our findings demonstrated the expression ratio of NaCh genes in normal fetal rat forebrain neurons to be Nav1.2>Nav1.3>Nav1.8>Nav1.1>Nav1.7 (rBII>rBIII >PN3>rBI>PN1). Veratridine treatment of neuronal cells produced neurotoxicity in a dose-dependent manner (0.25−20 μM). Neuronal injury caused by a dose of veratridine producing 80% cell death (2.5 μM) significantly, and exclusively down-regulated the Nav1.1 gene. However, treatment of neurons with RS100642 (200 μM) reversed the down-regulation of the Nav1.1. gene expression caused by veratridine. Our findings document for the first time quantitative and relative changes in the expression of various NaCh genes in neurons following injury produced by selective activation of voltage-gated sodium channels, and suggest that the Nav1.1 sodium channel gene may play a key role in the neuronal injury/recovery process.