Gregory D. Ford

Extended Therapeutic Window and Functional Recovery after Intraarterial Administration of Neuregulin-1 after Focal Ischemic Stroke

We have previously shown that neuregulin-1 (NRG-1) protects neurons from ischemic brain injury if administered before focal stroke. Here, we examined the therapeutic window and functional recovery after NRG-1 treatment in rats subjected to 90 mins of middle cerebral artery occlusion (MCAO) and 24 h of reperfusion. Neuregulin-1 (2.5 ng/kg bolus, 1.25 ng/kg/min infusion) reduced infarct volume by 89.2% ± 41.9% (mean ± s.d.; n = 8; P < 0.01) if administered immediately after the onset of reperfusion. Neuroprotection was also evident if NRG-1 was administered 4 h (66.4% ± 52.6%; n = 7; P < 0.01) and 12 h (57.0% ± 20.8%; n = 8; P < 0.01) after reperfusion. Neuregulin-1 administration also resulted in a significant improvement of functional neurologic outcome compared with vehicle-treated animals (32.1% ± 5.7%; n 9; P < 0.01). The neuroprotective effect of the single administration of NRG-1 was seen as long as 2 weeks after treatment. Neurons labeled with the neurodegeneration marker dye Fluoro-JadeB were observed after MCAO in the cortex, but the numbers were significantly reduced after NRG-1 treatment. These results indicate that NRG-1 is a potent neuroprotective compound with an extended therapeutic window that has practical therapeutic potential in treating individuals after ischemic brain injury.

Neuroprotection by neuregulin-1 in a rat model of permanent focal cerebral ischemia

Neuregulin-1 (NRG-1) is a growth factor with potent neuroprotective capacity in ischemic stroke. We recently showed that NRG-1 reduced neuronal death following transient middle cerebral artery occlusion (tMCAO) by up to 90% with an extended therapeutic window. Here, we examined the neuroprotective potential of NRG-1 using a permanent MCAO ischemia (pMCAO) rat model. NRG-1 reduced infarction in pMCAO by 50% when administered prior to ischemia. We previously demonstrated using gene expression profiling that pMCAO was associated with an exaggerated excitotoxicity response compared to tMCAO. Therefore, we examined whether co-treatment with an inhibitor of excitotoxicity would augment the effect of NRG-1 following pMCAO. Both NRG-1 and the N-methyl-D-aspartate (NMDA) antagonist MK-801 similarly reduced infarct size following pMCAO. However, combination treatment with both NRG-1 and MK-801 resulted in greater neuroprotection than either compound alone, including a 75% reduction in cortical infarction compared to control. Consistent with these findings, NRG-1 reduced neuronal death using an in vitro ischemia model and this effect was augmented by MK-801. These results demonstrate the efficacy of NRG-1 in pMCAO rat focal ischemia model. Our findings further indicate the potential clinically relevance of NRG-1 alone or as a combination strategy for treating ischemic stroke.

Spatiotemporal pattern of neuronal injury induced by DFP in rats: A model for delayed neuronal cell death following acute OP intoxication

Organophosphate (OP) neurotoxins cause acute cholinergic toxicity and seizures resulting in delayed brain damage and persistent neurological symptoms. Testing novel strategies for protecting against delayed effects of acute OP intoxication has been hampered by the lack of appropriate animal models. In this study, we characterize the spatiotemporal pattern of cellular injury after acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague-Dawley rats received pyridostigmine (0.1 mg/kg, im) and atropine methylnitrate (20mg/kg, im) prior to DFP (9 mg/kg, ip) administration. All DFP-treated animals exhibited moderate to severe seizures within minutes after DFP injection but survived up to 72 h. AChE activity was significantly depressed in the cortex, hippocampus, subcortical brain tissue and cerebellum at 1h post-DFP injection and this inhibition persisted for up to 72 h. Analysis of neuronal injury by Fluoro-Jade B (FJB) labeling revealed delayed neuronal cell death in the hippocampus, cortex, amygdala and thalamus, but not the cerebellum, starting at 4h and persisting until 72 h after DFP treatment, although temporal profiles varied between brain regions. At 24h post-DFP injection, the pattern of FJB labeling corresponded to TUNEL staining in most brain regions, and FJB-positive cells displayed reduced NeuN immunoreactivity but were not immunopositive for astrocytic (GFAP), oligodendroglial (O4) or macrophage/microglial (ED1) markers, demonstrating that DFP causes a region-specific delayed neuronal injury mediated in part by apoptosis. These findings indicate the feasibility of this model for testing neuroprotective strategies, and provide insight regarding therapeutic windows for effective pharmacological intervention following acute OP intoxication.

Expression Analysis Systematic Explorer (EASE) analysis reveals differential gene expression in permanent and transient focal stroke rat models.

To gain greater insight on the molecular mechanisms that underlie ischemic stroke, we compared gene expression profiles in transient (tMCAO) and permanent middle cerebral artery occlusion (pMCAO) stroke models using Expression Analysis Systematic Explorer (EASE) pathway analysis software. Many transcripts were induced in both stroke models, including genes associated with transcriptional pathways, cell death, stress responses and metabolism. However, EASE analysis of the regulated genes indicated molecular functions and biological processes unique to each model. Pathways associated with tMCAO included inflammation, apoptosis and cell cycle, while pMCAO was associated with the induction of genes encoding neurotransmitter receptors, ion channels, growth factors and signaling molecules. An intriguing finding was the involvement of tyrosine kinases and phosphatases following pMCAO. These results provide evidence that neuronal death following tMCAO and pMCAO involves distinct mechanisms. These findings may give new insight to the molecular mechanisms involved in stroke and may lead to novel neuroprotective strategies.

Neuroprotective effects of Neuregulin-1 on B35 Neuronal Cells following Ischemia

We previously showed that neuregulin-1 (NRG-1) protected neurons from death in vivo following focal ischemia. The goal of this study was to develop an in vitro rat ischemia model to examine the cellular and molecular mechanisms involved in the neuroprotective effects of NRG-1 on ischemia-induced neuronal death. Rat B-35 neuroblastoma cells differentiated by serum withdrawal, developed enhanced neuronal characteristics including, neurite extension and upregulation of neuronal markers of differentiation. When B35 neurons were subjected to oxygen glucose deprivation (OGD)/reoxygenation or glutamate, widespread neuronal death was seen after both treatments. Treatment with NRG-1 immediately after OGD significantly increased neuronal survival. NRG-1 administration also resulted in a significant decrease in annexin V, an early marker of apoptosis. However, the neurotoxic actions of glutamate were unaffected by NRG-1. The neuroprotective effects of NRG-1 were prevented by an inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. These results provide a new model to gain insight into the mechanisms employed by NRG-1 to protect neurons from ischemic brain injury.

Gene expression patterns following unilateral traumatic brain injury reveals a local pro-inflammatory and remote anti-inflammatory response

BackgroundTraumatic brain injury (TBI) results in irreversible damage at the site of impact and initiates cellular and molecular processes that lead to secondary neural injury in the surrounding tissue. We used microarray analysis to determine which genes, pathways and networks were significantly altered using a rat model of TBI. Adult rats received a unilateral controlled cortical impact (CCI) and were sacrificed 24 h post-injury. The ipsilateral hemi-brain tissue at the site of the injury, the corresponding contralateral hemi-brain tissue, and naïve (control) brain tissue were used for microarray analysis. Ingenuity Pathway Analysis (IPA) software was used to identify molecular pathways and networks that were associated with the altered gene expression in brain tissues following TBI.ResultsInspection of the top fifteen biological functions in IPA associated with TBI in the ipsilateral tissues revealed that all had an inflammatory component. IPA analysis also indicated that inflammatory genes were altered on the contralateral side, but many of the genes were inversely expressed compared to the ipsilateral side. The contralateral gene expression pattern suggests a remote anti-inflammatory molecular response. We created a network of the inversely expressed common (i.e., same gene changed on both sides of the brain) inflammatory response (IR) genes and those IR genes included in pathways and networks identified by IPA that changed on only one side. We ranked the genes by the number of direct connections each had in the network, creating a gene interaction hierarchy (GIH). Two well characterized signaling pathways, toll-like receptor/NF-kappaB signaling and JAK/STAT signaling, were prominent in our GIH.ConclusionsBioinformatic analysis of microarray data following TBI identified key molecular pathways and networks associated with neural injury following TBI. The GIH created here provides a starting point for investigating therapeutic targets in a ranked order that is somewhat different than what has been presented previously. In addition to being a vehicle for identifying potential targets for post-TBI therapeutic strategies, our findings can also provide a context for evaluating the potential of therapeutic agents currently in development.

Biomarkers for stroke

Background Major stroke clinical trials have failed during the past decades. The failures suggest the presence of heterogeneity among stroke patients. Biomarkers refer to indicators found in the blood, other body fluids or tissues that predicts physiologic or disease states, increased disease risk, or pharmacologic responses to a therapeutic intervention. Stroke biomarkers could be used as a guiding tool for more effective personalized therapy. Main Contents Three aspects of stroke biomarkers are explored in detail. First, the possible role of biomarkers in patients with stroke is discussed. Second, the limitations of conventional biomarkers (especially protein biomarkers) in the area of stroke research are presented with the reasons. Lastly, various types of biomarkers including traditional and novel genetic, microvesicle, and metabolomics-associated biomarkers are introduced with their advantages and disadvantages. We especially focus on the importance of comprehensive approaches using a variety of stroke biomarkers. Conclusion Although biomarkers are not recommended in practice guidelines for use in the diagnosis or treatment of stroke, many efforts have been made to overcome the limitations of biomarkers. The studies reviewed herein suggest that comprehensive analysis of different types of stroke biomarkers will improve the understanding of individual pathophysiologies and further promote the development of screening tools for of high-risk patients, and predicting models of stroke outcome and rational stroke therapy tailored to the characteristics of each case.

Neuregulin-1 is Neuroprotective in a Rat Model of Organophosphate-Induced Delayed Neuronal Injury

Current medical countermeasures against organophosphate (OP) nerve agents are effective in reducing mortality, but do not sufficiently protect the CNS from delayed brain damage and persistent neurological symptoms. In this study, we examined the efficacy of neuregulin-1 (NRG-1) in protecting against delayed neuronal cell death following acute intoxication with the OP diisopropylflurophosphate (DFP). Adult male Sprague-Dawley rats were pretreated with pyridostigmine (0.1 mg/kg BW, i.m.) and atropine methylnitrate (20 mg/kg BW, i.m.) prior to DFP (9 mg/kg BW, i.p.) intoxication to increase survival and reduce peripheral signs of cholinergic toxicity but not prevent DFP-induced seizures or delayed neuronal injury. Pretreatment with NRG-1 did not protect against seizures in rats exposed to DFP. However, neuronal injury was significantly reduced in most brain regions by pretreatment with NRG-1 isoforms NRG-EGF (3.2 μg/kg BW, i.a) or NRG-GGF2 (48 μg/kg BW, i.a.) as determined by FluroJade-B labeling in multiple brain regions at 24 h post-DFP injection. NRG-1 also blocked apoptosis and oxidative stress-mediated protein damage in the brains of DFP-intoxicated rats. Administration of NRG-1 at 1h after DFP injection similarly provided significant neuroprotection against delayed neuronal injury. These findings identify NRG-1 as a promising adjuvant therapy to current medical countermeasures for enhancing neuroprotection against acute OP intoxication.

Computational identification of conserved transcription factor binding sites upstream of genes induced in rat brain by transient focal ischemic stroke

Microarray analysis has been used to understand how gene regulation plays a critical role in neuronal injury, survival and repair following ischemic stroke. To identify the transcriptional regulatory elements responsible for ischemia-induced gene expression, we examined gene expression profiles of rat brains following focal ischemia and performed computational analysis of consensus transcription factor binding sites (TFBS) in the genes of the dataset. In this study, rats were sacrificed 24 h after middle cerebral artery occlusion (MCAO) stroke and gene transcription in brain tissues following ischemia/reperfusion was examined using Affymetrix GeneChip technology. The CONserved transcription FACtor binding site (CONFAC) software package was used to identify over-represented TFBS in the upstream promoter regions of ischemia-induced genes compared to control datasets. CONFAC identified 12 TFBS that were statistically over-represented from our dataset of ischemia-induced genes, including three members of the Ets-1 family of transcription factors (TFs). Microarray results showed that mRNA for Ets-1 was increased following tMCAO but not pMCAO. Immunohistochemical analysis of Ets-1 protein in rat brains following MCAO showed that Ets-1 was highly expressed in neurons in the brain of sham control animals. Ets-1 protein expression was virtually abolished in injured neurons of the ischemic brain but was unchanged in peri-infarct brain areas. These data indicate that TFs, including Ets-1, may influence neuronal injury following ischemia. These findings could provide important insights into the mechanisms that lead to brain injury and could provide avenues for the development of novel therapies.