Browsing by Autor "Alex Dang"

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Abstract TP102: Modulation of Neuroinflammation by Haptoglobin Reduces Oxidative Stress and Improves ICH Outcomes
(Lippincott Williams & Wilkins, 2016) Jenna L Leclerc; Alex Dang; Juan Santiago‐Moreno; Sylvain Doré
Intracerebral hemorrhage (ICH) is a stroke subtype associated with high morbidity and mortality. With breakdown of the blood-brain barrier and entry of toxic blood components and metabolites within the brain, a highly oxidative environment ensues and leads to a toxic neuroinflammatory cascade. A major cause of the debilitation following brain hemorrhage is due to the direct toxicity of blood components, notably hemoglobin (Hb), the most upstream precipitating factor in the cascade. The acute phase plasma protein haptoglobin (Hp) binds Hb and inhibits its cytotoxic, pro-oxidative, and pro-inflammatory properties. In this study, we investigated whether the local and specific overexpression of Hp would aid in the safe detoxification and clearance of free Hb, thereby protecting the neuropil from Hb-mediated oxidative stress and improving ICH outcomes. Hp was overexpressed locally within the brain using uniquely designed adeno-associated viral vectors and ICH was induced using the intrastriatal autologous whole blood injection model. Functional outcomes were assessed by a 24-point neurological deficit score. At 72h post-hemorrhage, mice were sacrificed and brains collected for histological staining. Hp-overexpressing mice demonstrated smaller lesion volumes (p<0.05) with less blood accumulation (p<0.05) and improve neurologic status after ICH (p<0.05) when compared to an identically treated control group (n=11-13/group). Histological staining for Iba-1, GFAP, heme oxygenase-1, 4-hydroxynonenal, ferric iron, and myeloperoxidase was performed and revealed: 1) significantly less heme oxygenase-1 expression and lipid peroxidation, 2) a trend towards reduced peripheral neutrophil infiltration, 3) significantly increased cortical microgliosis and cortical and striatal astrogliosis, and 4) no changes in ferric iron content or striatal microgliosis. In conclusion, Hp overexpression in the brain reduces ICH-induced brain injury and improves functional outcomes. Locally modulating brain Hp levels could represent an important clinically relevant strategy for the treatment of ICH.
Abstract W P237: Overexpression of Soluble Hemopexin as a Therapeutic Tool against Intracerebral Hemorrhage
(Lippincott Williams & Wilkins, 2015) Jenna L Leclerc; Alex Dang; Juan Santiago‐Moreno; Sylvain Doré
Intracerebral hemorrhage (ICH) has the highest morbidity, disability, and mortality rates of any stroke subtype, including ischemic stroke. When a hemorrhagic stroke occurs, the blood-brain barrier is broken and blood components enter the brain. A major cause of morbidity and mortality following ICH is the direct toxicity of blood metabolites, mainly free heme, on adjacent brain tissue. Hemopexin (Hpx) is the endogenous protein responsible for scavenging free heme; thereby, modulating its proxidant and proinflammatory properties. We hypothesized that the specific local overexpression of Hpx within the brain would attenuate anatomical and functional outcomes after experimentally inducing an ICH. We uniquely designed novel adeno-associated viral vectors (and control vectors) to specifically overexpress Hpx locally within the brain of C57BL/6N mice. After experimentally inducing an ICH using the autologous whole blood model, when compared to an identically treated control group, Hpx-overexpressing mice were found to have reduced brain injury volume at 72h post-ICH (p<0.05), as identified by lesion volume quantification of Cresyl Violet stained brain sections (n=7-9/group). Further, these anatomical outcomes were correlated with improved neurologic recovery, as measured by a 24-point neurological deficit scale, at 24h, 48h, and 72h post-ICH (p<0.05). Modulation of hemopexin levels would represent a clinically relevant strategy for the treatment of secondary brain injury following ICH. Further studies are needed in order to evaluate the in vivo utility of hemopexin in attenuating oxidative damage following hemorrhagic brain injury.
Abstract WMP106: Haptoglobin is Present as Zonulin in the Brain and Overexpression Improves Intracerebral Hemorrhage Outcomes
(Lippincott Williams & Wilkins, 2019) Jenna L Leclerc; Andrew S Lampert; Harrison Phillips; Tina Esfandiary; Alex Dang; Juan Santiago‐Moreno; Sylvain Doré
Intracerebral hemorrhage (ICH) most commonly results from a hypertensive spontaneous bleed into the brain parenchyma and is a devastating stroke subtype. Hemolysis ensues and large quantities of hemoglobin (Hb) are released that initiate a toxic oxidative and neuroinflammatory cascade. Hp is an abundant plasma glycoprotein that binds and detoxifies extracorpuscular Hb with strong affinity, although very little Hp is made endogenously within the brain. Of note, Hp undergoes a complex biosynthesis into a single polypeptide prohaptoglobin that is co-translationally processed in the endoplasmic reticulum to form the α- and β-subunits, which subsequently dimerize. We hypothesized that overexpressing Hp locally in the brain would lead to improved outcomes following ICH. Overexpression was achieved using adeno-associated viral vectors and ICH was induced using the intrastriatal autologous whole blood injection model. Functional outcomes were assessed by a 24-point neurological deficit score. Western blotting under reducing and non-reducing conditions and mass spectrometry were utilized to confirm the identity of the overexpression product, which surprisingly was prohaptoglobin, also known as zonulin. At 72h post-ICH, zonulin-overexpressing mice have smaller lesion volumes with less blood accumulation and better neurologic function (p<0.05) when compared to controls (n=11-13/group). Zonulin-overexpressing mice also demonstrate less heme oxygenase-1 expression, lipid peroxidation, and peripheral neutrophil infiltration, and significantly increased microgliosis and astrogliosis. No differences in iron content were seen. Importantly, these results provide evidence that Hp in its fully processed form is not made endogenously within the brain, but instead transcription from this gene results in the formation of zonulin, which has very distinctly different functions. Notably, zonulin does not bind Hb or provide redox protection, but is a strong regulator of intercellular tight junctions, which has important implications for intracranial bleeding. Interestingly, we show that zonulin-overexpression improves ICH outcomes and thus this pathway represents a new potentially targetable avenue for the treatment of ICH.
Abstract WP99: Specific and Local Overexpression of Hemopexin Improves Anatomical and Functional Outcomes in the Autologous Blood Intracerebral Hemorrhage Model
(Lippincott Williams & Wilkins, 2016) Jenna L Leclerc; Juan Santiago‐Moreno; Alex Dang; Sylvain Doré
Intracerebral hemorrhage (ICH) has the highest morbidity, disability, and mortality rates of any stroke subtype, including ischemic stroke. When a hemorrhagic stroke occurs, the blood-brain barrier is broken and blood components enter the brain. A major cause of morbidity and mortality following ICH is the direct toxicity of blood metabolites, notably free heme, on adjacent brain tissue. Hemopexin (Hpx) is the endogenous protein responsible for scavenging free heme; thereby, modulating its proxidant and proinflammatory properties. Given the low relative level of Hpx expression in the brain, we hypothesized that overexpression of Hpx would improve anatomical and functional outcomes after ICH. Unique adeno-associated viral vectors (and control vectors) were designed to specifically overexpress Hpx-(tags) locally within the brain. Hpx-overexpressing mice have smaller lesion volumes (p<0.05) and reduced blood accumulation (p<0.05) at 72h post-ICH (n=12-13/group). Improved anatomical outcomes are also associated with better neurologic functional recovery as measured by a 24-point neurological deficit scale (p<0.05). Histological staining for ferric iron, 4-hydroxynonenal, GFAP, Iba-1, heme oxygenase-1, and myeloperoxidase was performed and the distribution of Hpx-tag proteins was evaluated by Western blotting with anti-tag probing of brain homogenates, cerebrospinal fluid, and serum. Hpx-overexpressing mice have significantly increased cortical microgliosis and astrogliosis and peripheral neutrophil infiltration, but no changes in striatal microgliosis and astrogliosis, ferric iron content, heme oxygenase-1 expression, or lipid peroxidation. Additionally, Hpx-tag proteins were highly expressed in all three types of biological specimens evaluated. The increased levels of Hpx in the serum of Hpx-overexpressing mice were confirmed to be 62% higher by ELISA (p<0.01). These results indicate that the local overexpression of Hpx in the brain results in the capacity to facilitate the clearance of heme likely by central and peripheral mechanisms. Specific modulation of local Hpx levels may represent a clinically relevant strategy for the treatment of secondary brain injury following ICH.