Denis E. Bragin

In Vivo Inhibition of miR-155 Promotes Recovery after Experimental Mouse Stroke

A multifunctional microRNA, miR-155, has been recently recognized as an important modulator of numerous biological processes. In our previous in vitro studies, miR-155 was identified as a potential regulator of the endothelial morphogenesis. The present study demonstrates that in vivo inhibition of miR-155 supports cerebral vasculature after experimental stroke. Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Microvasculature in peri-infarct area, infarct size, and animal functional recovery were assessed at 1, 2, and 3 weeks after dMCAO. Using in vivo two-photon microscopy, we detected improved blood flow and microvascular integrity in the peri-infarct area of miR-155 inhibitor-injected mice. Electron microscopy revealed that, in contrast to the control group, these animals demonstrated well preserved capillary tight junctions (TJs). Western blot analysis data indicate that improved TJ integrity in the inhibitor-injected animals could be associated with stabilization of the TJ protein ZO-1 and mediated by the miR-155 target protein Rheb. MRI analysis showed significant (34%) reduction of infarct size in miR-155 inhibitor-injected animals at 21 d after dMCAO. Reduced brain injury was confirmed by electron microscopy demonstrating decreased neuronal damage in the peri-infarct area of stroke. Preservation of brain tissue was reflected in efficient functional recovery of inhibitor-injected animals. Based on our findings, we propose that in vivo miR-155 inhibition after ischemia supports brain microvasculature, reduces brain tissue damage, and improves the animal functional recovery. SIGNIFICANCE STATEMENT In the present study, we investigated an effect of the in vivo inhibition of a microRNA, miR-155, on brain recovery after experimental cerebral ischemia. To our knowledge, this is the first report describing the efficiency of intravenous anti-miRNA injections in a mouse model of ischemic stroke. The role of miRNAs in poststroke revascularization has been unexplored and in vivo regulation of miRNAs during the subacute phase of stroke has not yet been proposed. Our investigation introduces a new and unexplored approach to cerebral regeneration: regulation of poststroke angiogenesis and recovery through direct modulation of specific miRNA activity. We expect that our findings will lead to the development of novel strategies for regulating neurorestorative processes in the postischemic brain.

Development of epileptiform excitability in the deep entorhinal cortex after status epilepticus

Epileptiform neuronal activity during seizures is observed in many brain areas, but its origins following status epilepticus (SE) are unclear. We have used the Li low‐dose pilocarpine rat model of temporal lobe epilepsy to examine early development of epileptiform activity in the deep entorhinal cortex (EC). We show that during the 3‐week latent period that follows SE, an increasing percentage of neurons in EC layer 5 respond to a single synaptic stimulus with polysynaptic burst depolarizations. This change is paralleled by a progressive depolarizing shift of the inhibitory postsynaptic potential reversal potential in layer 5 neurons, apparently caused by upregulation of the Cl− inward transporter NKCC1 and concurrent downregulation of the Cl− outward transporter KCC2, both changes favoring intracellular Cl− accumulation. Inhibiting Cl− uptake in the latent period restored more negative GABAergic reversal potentials and eliminated polysynaptic bursts. The changes in the Cl− transporters were highly specific to the deep EC. They did not occur in layers 1–3, perirhinal cortex, subiculum or dentate gyrus during this period. We propose that the changes in Cl− homeostasis facilitate hyperexcitability in the deep entorhinal cortex leading to epileptiform discharge there, which subsequently affects downstream cortical regions.

Involvement of adenylate cyclase and tyrosine kinase signaling pathways in response of crayfish stretch receptor neuron and satellite glia cell to photodynamic treatment

Neuroglial interactions are most profound during development or damage of nerve tissue. We studied the responses of crayfish stretch receptor neurons (SRN) and satellite glial cells to photosensitization with sulfonated aluminum phthalocyanine Photosens. Although Photosens was localized mainly in the glial envelope, neurons were very sensitive to photodynamic treatment. Photosensitization gradually inhibited and then abolished neuron activity. Neuronal and glial nuclei shrank. Some neurons and glial cells lost the integrity of the plasma membrane and died through necrosis after the treatment. The nuclei of other glial cells but not neurons become fragmented, indicating apoptosis. The number of glial nuclei around neuron soma increased, probably indicating proliferation for enhanced neuron protection. Adenylate cyclase (AC) inhibition by MDL‐12330A, or tyrosine kinase (TK) inhibition by genistein, shortened neuron lifetime, whereas AC activation by forskolin or protein tyrosine phosphatases (PTP) inhibition by sodium orthovanadate prolonged neuronal activity. Therefore, cAMP and phosphotyrosines produced by AC and TK, respectively, protected SRN against photoinactivation. AC inhibition reduced photodamage of the plasma membrane and subsequent necrosis in neuronal and glial cells. AC activation prevented apoptosis in photosensitized glial cells and stimulated glial proliferation. TK inhibition protected neurons but not glia against photoinduced membrane permeabilization and subsequent necrosis whereas PTP inhibition more strongly protected glial cells. Therefore, both signaling pathways involving cAMP and phosphotyrosines might contribute to the maintenance of neuronal activity and the integrity of the neuronal and glial plasma membranes. Adenylate cyclase but not phosphotyrosine signaling pathways modulated glial apoptosis and proliferation under photooxidative stress.

Photodynamic Inactivation of Isolated Crayfish Mechanoreceptor Neuron: Different Death Modes Under Different Photosensitizer Concentrations¶

To study the mechanism of photodynamic nerve cell killing, isolated crayfish mechanoreceptor neurons were photosensitized by the sulfonated aluminum ophthalocyanine Photosens. Neuron activity was continuously recorded until irreversible abolition. Intense (10−5M Photosens) or weak (10−7M Photosens) photosensitization induced different bioelectric neuron responses: firing activation followed by irreversible depolarization block or gradual inhibition until firing abolition, respectively. These bioelectric responses were accompanied by different biochemical and morphological changes. In the case of intense photosensitization, neuron nuclei swelled and then shrank. Succinate dehydrogenase (SDH) was inhibited, and the plasma membrane was compromised just after firing cessation. Weak photosensitization did not induce these changes but caused swelling of the endoplasmic reticulum and destruction of the matrix, cristae and membranes in some of the mitochondria. Other mitochondria, however, retained the normal structure. Plasma membrane damage, SDH inhibition, nucleus shrinkage and impairment of the nuclear border occurred after 2–4 h. It is concluded that intense photosensitization induced necrotic processes during irradiation, whereas weaker impact caused delayed necrosis 2–4 h later. The observed electrophysiological neuron responses to photodynamic therapy may be considered as early hallmarks of different modes of forthcoming cell death.

High intracranial pressure effects on cerebral cortical microvascular flow in rats.

To manage patients with high intracranial pressure (ICP), clinicians need to know the critical cerebral perfusion pressure (CPP) required to maintain cerebral blood flow (CBF). Historically, the critical CPP obtained by decreasing mean arterial pressure (MAP) to lower CPP was 60 mm Hg, which fell to 30 mm Hg when CPP was reduced by increasing ICP. We examined whether this decrease in critical CPP was due to a pathological shift from capillary (CAP) to high-velocity microvessel flow or thoroughfare channel (TFC) shunt flow. Cortical microvessel red blood cell velocity and NADH fluorescence were measured by in vivo two-photon laser scanning microscopy in rats at CPP of 70, 50, and 30 mm Hg by increasing ICP or decreasing MAP. Water content was measured by wet/dry weight, and cortical perfusion by laser Doppler flux. Reduction of CPP by raising ICP increased TFC shunt flow from 30.4±2.3% to 51.2±5.2% (mean±SEM, p<0.001), NADH increased by 20.3±6.8% and 58.1±8.2% (p<0.01), and brain water content from 72.9±0.47% to 77.8±2.42% (p<0.01). Decreasing CPP by MAP decreased TFC shunt flow with a smaller rise in NADH and no edema. Doppler flux decreased less with increasing ICP than decreasing MAP. The decrease seen in the critical CPP with increased ICP is likely due to a redistribution of microvascular flow from capillary to microvascular shunt flow or TFC shunt flow, resulting in a pathologically elevated CBF associated with tissue hypoxia and brain edema, characteristic of non-nutritive shunt flow.

Differential changes of glutathione levels in astrocytes and neurons in ischemic brains by two-photon imaging

Using two-photon imaging techniques with monochlorobimane as a glutathione (GSH) probe, we investigated GSH levels in both core and penumbra regions of an ischemic brain after middle cerebral artery occlusion. We found that the GSH level significantly decreased in the ischemic core, but increased significantly in the penumbra. Furthermore, we observed a differential change of the GSH levels in neurons and astrocytes in the penumbra. The GSH level in neurons increased significantly whereas it decreased slightly in astrocytes in the penumbra. These findings reveal critical region and cell type-dependent changes of the GSH level in an ischemic brain.

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.

EFFECT OF CEREBRAL PERFUSION PRESSURE ON CEREBRAL CORTICAL MICROVASCULAR SHUNTING AT HIGH INTRACRANIAL PRESSURE IN RATS

Background and Purpose— Recently, we showed that decreasing cerebral perfusion pressure (CPP) from 70 mm Hg to 50 mm Hg and 30 mm Hg by increasing intracranial pressure (ICP) with a fluid reservoir induces a transition from capillary (CAP) to microvascular shunt (MVS) flow in the uninjured rat brain. This transition was associated with tissue hypoxia, increased blood–brain barrier (BBB) permeability, and brain edema. Our aim was to determine whether an increase in CPP would attenuate the transition to MVS flow at high ICP. Methods— Rats were subjected to progressive, step-wise increases in ICP of up to 60 mm Hg by an artificial cerebrospinal fluid reservoir connected to the cisterna magna. CPP was maintained at 50, 60, 70, or 80 mm Hg by intravenous dopamine infusion. Microvascular red blood cell flow velocity, BBB integrity (fluorescein dye extravasation), and tissue oxygenation (nicotinamide adenine dinucleotide) were measured by in vivo 2-photon laser scanning microscopy. Doppler cortical flux, rectal and cranial temperatures, ICP, arterial blood pressure, and gases were monitored. Results— The CAP/MVS ratio increased (P<0.05) at higher ICP as CPP was increased from 50 to 80 mm Hg. At an ICP of 30 mm Hg and CPP of 50 mm Hg, the CAP/MVS ratio was 0.6±0.1. At CPP of 60, 70, and 80 mm Hg, the ratio increased to 0.9±0.1, 1.4±0.1, and 1.9±0.1, respectively (mean±SEM; P<0.05). BBB opening and increase of reduced form of nicotinamide adenine dinucleotide occurred at higher ICP as CPP was increased. Conclusions— Increasing CPP at high ICP attenuates the transition from CAP to MVS flow, development of tissue hypoxia, and increased BBB permeability.

Increases in microvascular perfusion and tissue oxygenation via pulsed electromagnetic fields in the healthy rat brain

OBJECT High-frequency pulsed electromagnetic field stimulation is an emerging noninvasive therapy being used clinically to facilitate bone and cutaneous wound healing. Although the mechanisms of action of pulsed electromagnetic fields (PEMF) are unknown, some studies suggest that its effects are mediated by increased nitric oxide (NO), a well-known vasodilator. The authors hypothesized that in the brain, PEMF increase NO, which induces vasodilation, enhances microvascular perfusion and tissue oxygenation, and may be a useful adjunct therapy in stroke and traumatic brain injury. To test this hypothesis, they studied the effect of PEMF on a healthy rat brain with and without NO synthase (NOS) inhibition. METHODS In vivo two-photon laser scanning microscopy (2PLSM) was used on the parietal cortex of rat brains to measure microvascular tone and red blood cell (RBC) flow velocity in microvessels with diameters ranging from 3 to 50 μm, which includes capillaries, arterioles, and venules. Tissue oxygenation (reduced nicotinamide adenine dinucleotide [NADH] fluorescence) was also measured before and for 3 hours after PEMF treatment using the FDA-cleared SofPulse device (Ivivi Health Sciences, LLC). To test NO involvement, the NOS inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) was intravenously injected (10 mg/kg). In a time control group, PEMF were not used. Doppler flux (0.8-mm probe diameter), brain and rectal temperatures, arterial blood pressure, blood gases, hematocrit, and electrolytes were monitored. RESULTS Pulsed electromagnetic field stimulation significantly dilated cerebral arterioles from a baseline average diameter of 26.4 ± 0.84 μm to 29.1 ± 0.91 μm (11 rats, p < 0.01). Increased blood volume flow through dilated arterioles enhanced capillary flow with an average increase in RBC flow velocity by 5.5% ± 1.3% (p < 0.01). Enhanced microvascular flow increased tissue oxygenation as reflected by a decrease in NADH autofluorescence to 94.7% ± 1.6% of baseline (p < 0.05). Nitric oxide synthase inhibition by L-NAME prevented PEMF-induced changes in arteriolar diameter, microvascular perfusion, and tissue oxygenation (7 rats). No changes in measured parameters were observed throughout the study in the untreated time controls (5 rats). CONCLUSIONS This is the first demonstration of the acute effects of PEMF on cerebral cortical microvascular perfusion and metabolism. Thirty minutes of PEMF treatment induced cerebral arteriolar dilation leading to an increase in microvascular blood flow and tissue oxygenation that persisted for at least 3 hours. The effects of PEMF were mediated by NO, as we have shown in NOS inhibition experiments. These results suggest that PEMF may be an effective treatment for patients after traumatic or ischemic brain injury. Studies on the effect of PEMF on the injured brain are in progress.

Critical Cerebral Perfusion Pressure at High Intracranial Pressure Measured by Induced Cerebrovascular and Intracranial Pressure Reactivity

Objectives:The lower limit of cerebral blood flow autoregulation is the critical cerebral perfusion pressure at which cerebral blood flow begins to fall. It is important that cerebral perfusion pressure be maintained above this level to ensure adequate cerebral blood flow, especially in patients with high intracranial pressure. However, the critical cerebral perfusion pressure of 50 mm Hg, obtained by decreasing mean arterial pressure, differs from the value of 30 mm Hg, obtained by increasing intracranial pressure, which we previously showed was due to microvascular shunt flow maintenance of a falsely high cerebral blood flow. The present study shows that the critical cerebral perfusion pressure, measured by increasing intracranial pressure to decrease cerebral perfusion pressure, is inaccurate but accurately determined by dopamine-induced dynamic intracranial pressure reactivity and cerebrovascular reactivity. Design:Cerebral perfusion pressure was decreased either by increasing intracranial pressure or decreasing mean arterial pressure and the critical cerebral perfusion pressure by both methods compared. Cortical Doppler flux, intracranial pressure, and mean arterial pressure were monitored throughout the study. At each cerebral perfusion pressure, we measured microvascular RBC flow velocity, blood-brain barrier integrity (transcapillary dye extravasation), and tissue oxygenation (reduced nicotinamide adenine dinucleotide) in the cerebral cortex of rats using in vivo two-photon laser scanning microscopy. Setting:University laboratory. Subjects:Male Sprague-Dawley rats. Interventions:At each cerebral perfusion pressure, dopamine-induced arterial pressure transients (~10 mm Hg, ~45 s duration) were used to measure induced intracranial pressure reactivity (&Dgr; intracranial pressure/&Dgr; mean arterial pressure) and induced cerebrovascular reactivity (&Dgr; cerebral blood flow/&Dgr; mean arterial pressure). Measurements and Main Results:At a normal cerebral perfusion pressure of 70 mm Hg, 10 mm Hg mean arterial pressure pulses had no effect on intracranial pressure or cerebral blood flow (induced intracranial pressure reactivity = –0.03 ± 0.07 and induced cerebrovascular reactivity = –0.02 ± 0.09), reflecting intact autoregulation. Decreasing cerebral perfusion pressure to 50 mm Hg by increasing intracranial pressure increased induced intracranial pressure reactivity and induced cerebrovascular reactivity to 0.24 ± 0.09 and 0.31 ± 0.13, respectively, reflecting impaired autoregulation (p < 0.05). By static cerebral blood flow, the first significant decrease in cerebral blood flow occurred at a cerebral perfusion pressure of 30 mm Hg (0.71 ± 0.08, p < 0.05). Conclusions:Critical cerebral perfusion pressure of 50 mm Hg was accurately determined by induced intracranial pressure reactivity and induced cerebrovascular reactivity, whereas the static method failed.