Olga Bragina

Glucose up-regulates HIF-1α expression in primary cortical neurons in response to hypoxia through maintaining cellular redox status

It has been suggested that hypoxia‐inducible factor 1 (HIF‐1), a key regulator in cell’s adaptation to hypoxia, plays an important role in the fate of neurons during ischemia. However, the mechanism of HIF‐1 regulation is still not fully understood in neurons subjected to ischemia. In this study, we demonstrated that glucose up‐regulated the expression of HIF‐1α, the oxygen‐dependent subunit of HIF‐1, in rat primary cortical neurons exposed to hypoxia. To understand the mechanism of glucose‐regulated HIF‐1α expression, we investigated the relationships between HIF‐1α expression, reactive oxygen species (ROS), and redox status. Low levels of HIF‐1α protein expression were observed in the neurons exposed to in vitro ischemic conditions that had high levels of ROS (oxidizing environments), and vice versa. The glutathione (GSH) precursor, N‐acetyl cysteine, induced HIF‐1α protein expression in hypoxic neurons while the GSH synthesis inhibitor, l‐buthionine sulfoximine, inhibited the expression. Moreover, (−)‐epicatechin gallate, a ROS scavenger, elevated HIF‐1α expression in the neurons subjected to in vitro ischemia. Furthermore, results from a systemic hypoxia model showed that a reducing environment increased HIF‐1α expression in rat brains. Taken together, these data presented the first evidence that glucose promoted HIF‐1α stabilization through regulating redox status in primary neurons exposed to hypoxia. The results imply that hypoxia only may not be sufficient to stabilize HIF‐1α and that a reducing environment is required to stabilize HIF‐1α in neurons exposed to hypoxia.

The role of microRNAs in neural stem cell-supported endothelial morphogenesis.

Functional signaling between neural stem/progenitor cells (NSPCs) and brain endothelial cells (ECs) is essential to the coordination of organized responses during initial embryonic development and also during tissue repair, which occurs following brain injury. In this study, we investigated the molecular mechanisms underlying this functional signaling, using primary mouse brain ECs and NSPCs from embryonic mouse brain. EC/NSPC co-culture experiments have revealed that neural progenitors secrete factors supporting angiogenesis, which induce noticeable changes in endothelial morphology. We demonstrate that NSPCs influence the expression of mTOR and TGF-β signaling pathway components implicated in the regulation of angiogenesis. Endothelial morphogenesis, an essential component of vascular development, is a complex process involving gene activation and the upregulation of specific cell signaling pathways. Recently identified small molecules, called microRNAs (miRNAs), regulate the expression of genes and proteins in many tissues, including brain and vasculature. We found that NSPCs induced considerable changes in the expression of at least 24 miRNAs and 13 genes in ECs. Three NSPC-regulated EC miRNAs were identified as the potential primary mediators of this NSPC/EC interaction. We found that the specific inhibition, or overexpression, of miRNAs miR-155, miR-100, and miR-let-7i subsequently altered the expression of major components of the mTOR, TGF-β and IGF-1R signaling pathways in ECs. Overexpression of these miRNAs in ECs suppressed, while inhibition activated, the in vitro formation of capillary-like structures, a process representative of EC morphogenesis. In addition, we demonstrate that inhibition of FGF, VEGF, and TGF-β receptor signaling abolished NSPC-promoted changes in the endothelial miRNA profiles. Our findings demonstrate that NSPCs induce changes in the miRNA expression of ECs, which are capable of activating angiogenesis by modulating distinct cell signaling pathways.

Enhancement of neurogenesis and memory by a neurotrophic peptide in mild to moderate traumatic brain injury.

BACKGROUND Traumatic brain injury (TBI) is a risk factor for Alzheimer disease (AD), a neurocognitive disorder with similar cellular abnormalities. We recently discovered a small molecule (Peptide 6) corresponding to an active region of human ciliary neurotrophic factor, with neurogenic and neurotrophic properties in mouse models of AD and Down syndrome. OBJECTIVE To describe hippocampal abnormalities in a mouse model of mild to moderate TBI and their reversal by Peptide 6. METHODS TBI was induced in adult C57Bl6 mice using controlled cortical impact with 1.5 mm of cortical penetration. The animals were treated with 50 nmol/d of Peptide 6 or saline solution for 30 days. Dentate gyrus neurogenesis, dendritic and synaptic density, and AD biomarkers were quantitatively analyzed, and behavioral tests were performed. RESULTS Ipsilateral neuronal loss in CA1 and the parietal cortex and increase in Alzheimer-type hyperphosphorylated tau and A-β were seen in TBI mice. Compared with saline solution, Peptide 6 treatment increased the number of newborn neurons, but not uncommitted progenitor cells, in dentate gyrus by 80%. Peptide 6 treatment also reversed TBI-induced dendritic and synaptic density loss while increasing activity in tri-synaptic hippocampal circuitry, ultimately leading to improvement in memory recall on behavioral testing. CONCLUSION Long-term treatment with Peptide 6 enhances the pool of newborn neurons in the dentate gyrus, prevents neuronal loss in CA1 and parietal cortex, preserves the dendritic and synaptic architecture in the hippocampus, and improves performance on a hippocampus-dependent memory task in TBI mice. These findings necessitate further inquiry into the therapeutic potential of small molecules based on neurotrophic factors.

High Intracranial Pressure Induced Injury in the Healthy Rat Brain.

Objectives:We recently showed that increased intracranial pressure to 50 mm Hg in the healthy rat brain results in microvascular shunt flow characterized by tissue hypoxia, edema, and increased blood-brain barrier permeability. We now determined whether increased intracranial pressure results in neuronal injury by Fluoro-Jade stain and whether changes in cerebral blood flow and cerebral metabolic rate for oxygen suggest nonnutritive microvascular shunt flow. Design:Intracranial pressure was elevated by a reservoir of artificial cerebrospinal fluid connected to the cisterna magna. Arterial blood gases, cerebral arterial-venous oxygen content difference, and cerebral blood flow by MRI were measured. Fluoro-Jade stain neurons were counted in histologic sections of the right and left dorsal and lateral cortices and hippocampus. Setting:University laboratory. Subjects:Male Sprague Dawley rats. Interventions:Arterial pressure support if needed by IV dopamine infusion and base deficit corrected by sodium bicarbonate. Measurements and Main Results:Fluoro-Jade stain neurons increased 2.5- and 5.5-fold at intracranial pressures of 30 and 50 mm Hg and cerebral perfusion pressures of 57 ± 4 (mean ± SEM) and 47 ± 6 mm Hg, respectively (p < 0.001) (highest in the right and left cortices). Voxel frequency histograms of cerebral blood flow showed a pattern consistent with microvascular shunt flow by dispersion to higher cerebral blood flow at high intracranial pressure and decreased cerebral metabolic rate for oxygen. Conclusions:High intracranial pressure likely caused neuronal injury because of a transition from normal capillary flow to nonnutritive microvascular shunt flow resulting in tissue hypoxia and edema, and it is manifest by a reduction in the cerebral metabolic rate for oxygen.

Rheological effects of drag-reducing polymers improve cerebral blood flow and oxygenation after traumatic brain injury in rats

Cerebral ischemia has been clearly demonstrated after traumatic brain injury (TBI); however, neuroprotective therapies have not focused on improvement of the cerebral microcirculation. Blood soluble drag-reducing polymers (DRP), prepared from high molecular weight polyethylene oxide, target impaired microvascular perfusion by altering the rheological properties of blood and, until our recent reports, has not been applied to the brain. We hypothesized that DRP improve cerebral microcirculation and oxygenation after TBI. DRP were studied in healthy and traumatized rat brains and compared to saline controls. Using in-vivo two-photon laser scanning microscopy over the parietal cortex, we showed that after TBI, nanomolar concentrations of intravascular DRP significantly enhanced microvascular perfusion and tissue oxygenation in peri-contusional areas, preserved blood–brain barrier integrity and protected neurons. The mechanisms of DRP effects were attributable to reduction of the near-vessel wall cell-free layer which increased near-wall blood flow velocity, microcirculatory volume flow, and number of erythrocytes entering capillaries, thereby reducing capillary stasis and tissue hypoxia as reflected by a reduction in NADH. Our results indicate that early reduction in CBF after TBI is mainly due to ischemia; however, metabolic depression of contused tissue could be also involved.

Application of optical coherence tomography for in vivo monitoring of the meningeal lymphatic vessels during opening of blood–brain barrier: mechanisms of brain clearing

Abstract. The meningeal lymphatic vessels were discovered 2 years ago as the drainage system involved in the mechanisms underlying the clearance of waste products from the brain. The blood–brain barrier (BBB) is a gatekeeper that strongly controls the movement of different molecules from the blood into the brain. We know the scenarios during the opening of the BBB, but there is extremely limited information on how the brain clears the substances that cross the BBB. Here, using the model of sound-induced opening of the BBB, we clearly show how the brain clears dextran after it crosses the BBB via the meningeal lymphatic vessels. We first demonstrate successful application of optical coherence tomography (OCT) for imaging of the lymphatic vessels in the meninges after opening of the BBB, which might be a new useful strategy for noninvasive analysis of lymphatic drainage in daily clinical practice. Also, we give information about the depth and size of the meningeal lymphatic vessels in mice. These new fundamental data with the applied focus on the OCT shed light on the mechanisms of brain clearance and the role of lymphatic drainage in these processes that could serve as an informative platform for a development of therapy and diagnostics of diseases associated with injuries of the BBB such as stroke, brain trauma, glioma, depression, or Alzheimer disease.

Drag-Reducing Polymer Enhances Microvascular Perfusion in the Traumatized Brain with Intracranial Hypertension

Current treatments for traumatic brain injury (TBI) have not focused on improving microvascular perfusion. Drag-reducing polymers (DRP), linear, long-chain, blood-soluble, nontoxic macromolecules, may offer a new approach to improving cerebral perfusion by primary alteration of the fluid dynamic properties of blood. Nanomolar concentrations of DRP have been shown to improve hemodynamics in animal models of ischemic myocardium and ischemic limb, but have not yet been studied in the brain. We recently demonstrated that DRP improved microvascular perfusion and tissue oxygenation in a normal rat brain. We hypothesized that DRP could restore microvascular perfusion in hypertensive brain after TBI. Using in vivo two-photon laser scanning microscopy we examined the effect of DRP on microvascular blood flow and tissue oxygenation in hypertensive rat brains with and without TBI. DRP enhanced and restored capillary flow, decreased microvascular shunt flow, and, as a result, reduced tissue hypoxia in both nontraumatized and traumatized rat brains at high intracranial pressure. Our study suggests that DRP could constitute an effective treatment for improving microvascular flow in brain ischemia caused by high intracranial pressure after TBI.

Pulsed Electromagnetic Field (PEMF) Mitigates High Intracranial Pressure (ICP) Induced Microvascular Shunting (MVS) in Rats

OBJECTIVE High-frequency pulsed electromagnetic field (PEMF) stimulation is an emerging noninvasive therapy that we have shown increases cerebral blood flow (CBF) and tissue oxygenation in the healthy rat brain. In this work, we tested the effect of PEMF on the brain at high intracranial pressure (ICP). We previously showed that high ICP in rats caused a transition from capillary (CAP) to non-nutritive microvascular shunt (MVS) flow, tissue hypoxia and increased blood brain barrier (BBB) permeability. METHODS Using in vivo two-photon laser scanning microscopy (2PLSM) over the rat parietal cortex, and studied the effects of PEMF on microvascular blood flow velocity, tissue oxygenation (NADH autofluorescence), BBB permeability and neuronal necrosis during 4 h of elevated ICP to 30 mmHg. RESULTS PEMF significantly dilated arterioles, increased capillary blood flow velocity and reduced MVS/capillary ratio compared to sham-treated animals. These effects led to a significant decrease in tissue hypoxia, BBB degradation and neuronal necrosis. CONCLUSIONS PEMF attenuates high ICP-induced pathological microcirculatory changes, tissue hypoxia, BBB degradation and neuronal necrosis.

Off-axis holographic laser speckle contrast imaging of blood vessels in tissues

Abstract. Laser speckle contrast imaging (LSCI) has become one of the most common tools for functional imaging in tissues. Incomplete theoretical description and sophisticated interpretation of measurement results are completely sidelined by a low-cost and simple hardware, fastness, consistent results, and repeatability. In addition to the relatively low measuring volume with around 700  μm of the probing depth for the visible spectral range of illumination, there is no depth selectivity in conventional LSCI configuration; furthermore, in a case of high NA objective, the actual penetration depth of light in tissues is greater than depth of field (DOF) of an imaging system. Thus, the information about these out-of-focus regions persists in the recorded frames but cannot be retrieved due to intensity-based registration method. We propose a simple modification of LSCI system based on the off-axis holography to introduce after-registration refocusing ability to overcome both depth-selectivity and DOF problems as well as to get the potential possibility of producing a cross-section view of the specimen.

Improvement of Impaired Cerebral Microcirculation Using Rheological Modulation by Drag-Reducing Polymers

Nanomolar intravascular concentrations of drag-reducing polymers (DRP) have been shown to improve hemodynamics and survival in animal models of ischemic myocardium and limb, but the effects of DRP on the cerebral microcirculation have not yet been studied. We recently demonstrated that DRP enhance microvascular flow in normal rat brain and hypothesized that it would restore impaired microvascular perfusion and improve outcomes after focal ischemia and traumatic brain injury (TBI). We studied the effects of DRP (high molecular weight polyethylene oxide, 4000 kDa, i.v. at 2 μg/mL of blood) on microcirculation of the rat brain: (1) after permanent middle cerebral artery occlusion (pMCAO); and (2) after TBI induced by fluid percussion. Using in vivo two-photon laser scanning microscopy (2PLSM) over the parietal cortex of anesthetized rats we showed that both pMCAO and TBI resulted in progressive decrease in microvascular circulation, leading to tissue hypoxia (NADH increase) and increased blood brain barrier (BBB) degradation. DRP, injected post insult, increased blood volume flow in arterioles and red blood cell (RBC) flow velocity in capillaries mitigating capillary stasis, tissue hypoxia and BBB degradation, which improved neuronal survival (Fluoro-Jade B, 24 h) and neurologic outcome (Rotarod, 1 week). Improved microvascular perfusion by DRP may be effective in the treatment of ischemic stroke and TBI.