Role of Pial Microvasospasms and Leukocyte Plugging for Parenchymal Perfusion after Subarachnoid Hemorrhage Assessed by In Vivo Multi-Photon Microscopy

Subarachnoid hemorrhage (SAH) is associated with acute and delayed cerebral ischemia. We suggested spasms of pial arterioles as a possible mechanism; however, it remained unclear whether and how pial microvasospasms (MVSs) induce cerebral ischemia. Therefore, we used in vivo deep tissue imaging by two-photon microscopy to investigate MVSs together with the intraparenchymal microcirculation in a clinically relevant murine SAH model. Male C57BL/6 mice received a cranial window. Cerebral vessels and leukocytes were labelled with fluorescent dyes and imaged by in vivo two-photon microscopy before and three hours after SAH induced by filament perforation. After SAH, a large clot formed around the perforation site at the skull base, and blood distributed along the perivascular space of the middle cerebral artery up to the cerebral cortex. Comparing the cerebral microvasculature before and after SAH, we identified three different patterns of constrictions: pearl string, global, and bottleneck. At the same time, the volume of perfused intraparenchymal vessels and blood flow velocity in individual arterioles were significantly reduced by more than 60%. Plugging of capillaries by leukocytes was observed but infrequent. The current study demonstrates that perivascular blood is associated with spasms of pial arterioles and that these spasms result in a significant reduction in cortical perfusion after SAH. Thus, the pial microvasospasm seems to be an important mechanism by which blood in the subarachnoid space triggers cerebral ischemia after SAH. Identifying the mechanisms of pial vasospasm may therefore result in novel therapeutic options for SAH patients.

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Long-term impairment of neurovascular coupling following experimental subarachnoid hemorrhage

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x19863021 ◽

2019 ◽

Vol 40 (6) ◽

pp. 1193-1202 ◽

Cited By ~ 2

Author(s):

Matilde Balbi ◽

Max Jativa Vega ◽

Athanasios Lourbopoulos ◽

Nicole A Terpolilli ◽

Nikolaus Plesnila

Keyword(s):

Subarachnoid Hemorrhage ◽

Delayed Cerebral Ischemia ◽

Neurovascular Coupling ◽

Early Brain Injury ◽

Vessel Diameter ◽

Two Photon Microscopy ◽

Two Photon ◽

Experimental Subarachnoid Hemorrhage

CO2-reactivity and neurovascular coupling are sequentially lost within the first 24 h after subarachnoid hemorrhage (SAH). Whether and when these impairments recover is not known. Therefore, we investigated the reactivity of pial and intraparenchymal vessels by in vivo two-photon microscopy one month after experimental SAH. C57BL/6 mice were subjected to either sham surgery or SAH by filament perforation. One month later, cerebral blood flow following CO2-challenge and forepaw stimulation was assessed by laser Doppler fluxmetry. Diameters of pial and intraparenchymal arterioles were quantified by in vivo two-photon microscopy. One month after SAH, pial and parenchymal vessels dilated in response to CO2. Neurovascular coupling was almost completely absent after SAH: vessel diameter did not change upon forepaw stimulation compared to a 20% increase in sham-operated mice. The current results demonstrate that neurovascular function differentially recovers after SAH: while CO2-reactivity normalizes within one month after SAH, neurovascular coupling is still absent. These findings show an acute and persistent loss of neurovascular coupling after SAH that may serve as a link between early brain injury and delayed cerebral ischemia, two distinct pathophysiological phenomena after SAH that were so far believed not to be directly related.

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In vivo imaging of activated microglia in a mouse model of focal cerebral ischemia by two-photon microscopy

Biomedical Optics Express ◽

10.1364/boe.6.003303 ◽

2015 ◽

Vol 6 (9) ◽

pp. 3303 ◽

Cited By ~ 10

Author(s):

Seoyeon Bok ◽

Taejun Wang ◽

Chan-Ju Lee ◽

Seong-Uk Jeon ◽

Young-Eun Kim ◽

...

Keyword(s):

Cerebral Ischemia ◽

Mouse Model ◽

In Vivo Imaging ◽

Focal Cerebral Ischemia ◽

Activated Microglia ◽

Two Photon Microscopy ◽

Two Photon

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MEK1/2 Inhibitor U0126 but Not Endothelin Receptor Antagonist Clazosentan Reduces Upregulation of Cerebrovascular Contractile Receptors and Delayed Cerebral Ischemia, and Improves Outcome after Subarachnoid Hemorrhage in Rats

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2014.205 ◽

2014 ◽

Vol 35 (2) ◽

pp. 329-337 ◽

Cited By ~ 13

Author(s):

Gro K Povlsen ◽

Lars Edvinsson

Keyword(s):

Subarachnoid Hemorrhage ◽

Cerebral Ischemia ◽

Cerebral Arteries ◽

Delayed Cerebral Ischemia ◽

Receptor Activation ◽

Receptor Subtypes ◽

Endothelin Receptor ◽

Elevated Expression ◽

Causes Of Mortality

Cerebral vasospasm and late cerebral ischemia (LCI) remain leading causes of mortality in patients experiencing a subarachnoid hemorrhage (SAH). This occurs typically 3 to 4 days after the initial bleeding and peaks at 5 to 7 days. The underlying pathophysiology is still poorly understood. Because SAH is associated with elevated levels of endothelin-1 (ET-1), focus has been on counteracting endothelin receptor activation with receptor antagonists like clazosentan, however, with poor outcome in clinical trials. We hypothesize that inhibition of intracellular transcription signaling will be an effective approach to prevent LCI. Here, we compare the effects of clazosentan versus the MEK1/2 blocker U0126 in a rat model of SAH. Although clazosentan directly inhibits the contractile responses in vivo to ET-1, it did not prevent SAH-induced upregulation of ET receptors in cerebral arteries and did not show a beneficial effect on neurologic outcome. U0126 had no vasomotor effect by itself but counteracts SAH-induced receptor upregulation in cerebral arteries and improved outcome after SAH. We suggest that because SAH induces elevated expression of several contractile receptor subtypes, it is not sufficient to block only one of these (ET receptors) but inhibition of transcriptional MEK1/2-mediated upregulation of several contractile receptors may be a viable way towards alleviating LCI.

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Acute changes in neurovascular reactivity after subarachnoid hemorrhage in vivo

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x15621253 ◽

2016 ◽

Vol 37 (1) ◽

pp. 178-187 ◽

Cited By ~ 9

Author(s):

Matilde Balbi ◽

Masayo Koide ◽

Susanne M Schwarzmaier ◽

George C Wellman ◽

Nikolaus Plesnila

Keyword(s):

Subarachnoid Hemorrhage ◽

Neuronal Damage ◽

Brain Slices ◽

Neurovascular Coupling ◽

Differential Interference Contrast Microscopy ◽

Two Photon Microscopy ◽

Two Photon ◽

Normal Increase ◽

Acute Changes

Subarachnoid hemorrhage causes acute and long-lasting constrictions of pial arterioles. Whether these vessels dilate normally to neuronal activity is of great interest since a mismatch between delivery and consumption of glucose and oxygen may cause additional neuronal damage. Therefore, we investigated neurovascular reactivity of pial and parenchymal arterioles after experimental subarachnoid hemorrhage. C57BL/6 mice were subjected to subarachnoid hemorrhage by filament perforation or sham surgery. Neurovascular reactivity was assessed 3 h later by forepaw stimulation or inhalation of 10% CO2. Diameters of cerebral arterioles were assessed using two-photon microscopy. Neurovascular coupling and astrocytic endfoot Ca2+ were measured in brain slices using two-photon and infrared-differential interference contrast microscopy. Vessels of sham-operated mice dilated normally to CO2 and forepaw stimulation. Three hours after subarachnoid hemorrhage, CO2 reactivity was completely lost in both pial and parenchymal arterioles, while neurovascular coupling was not affected. Brain slices studies also showed normal neurovascular coupling and a normal increase in astrocytic endfoot Ca2+ acutely after subarachnoid hemorrhage. These findings suggest that communication between neurons, astrocytes, and parenchymal arterioles is not affected in the first few hours after subarachnoid hemorrhage, while CO2 reactivity, which is dependent on NO signaling, is completely lost.

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Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1514209112 ◽

2015 ◽

Vol 112 (36) ◽

pp. 11377-11382 ◽

Cited By ~ 65

Author(s):

Carsten Tischbirek ◽

Antje Birkner ◽

Hongbo Jia ◽

Bert Sakmann ◽

Arthur Konnerth

Keyword(s):

Action Potential ◽

Functional Imaging ◽

Fluorescent Dyes ◽

Signal To Noise Ratio ◽

Single Action ◽

Two Photon Microscopy ◽

Two Photon ◽

Mouse Cortex ◽

Good Signal

In vivo Ca2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Ca2+ (Kd = 561 nM), single-action potential-evoked Ca2+ transients were discernable in most neurons with a good signal-to-noise ratio. Action potential-dependent Ca2+ transients were recorded in neurons of all six layers of the cortex at depths of up to −900 µm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca2+ indicators. Ca2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca2+ indicator Cal-590 is well suited for in vivo two-photon Ca2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.

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in vivo 3D Morphology of Astrocyte—Vasculature Interactions in the Somatosensory Cortex: Implications for Neurovascular Coupling

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2010.204 ◽

2010 ◽

Vol 31 (3) ◽

pp. 795-806 ◽

Cited By ~ 90

Author(s):

Addason F H McCaslin ◽

Brenda R Chen ◽

Andrew J Radosevich ◽

Bruno Cauli ◽

Elizabeth M C Hillman

Keyword(s):

Blood Vessels ◽

Somatosensory Cortex ◽

Three Dimensional ◽

Neurovascular Coupling ◽

In Vivo Studies ◽

Two Photon Microscopy ◽

Two Photon ◽

Pial Arterioles ◽

Capillary Bed

Astrocytes are increasingly believed to play an important role in neurovascular coupling. Recent in vivo studies have shown that intracellular calcium levels in astrocytes correlate with reactivity in adjacent diving arterioles. However, the hemodynamic response to stimulation involves a complex orchestration of vessel dilations and constrictions that spread rapidly over wide distances. In this work, we study the three-dimensional cytoarchitecture of astrocytes and their interrelations with blood vessels down through layer IV of the mouse somatosensory cortex using in vivo two-photon microscopy. Vessels and astrocytes were visualized through intravenous dextran-conjugated fluorescein and cortically applied sulforhodamine 101 (SR101), respectively. In addition to exploring astrocyte density, vascular proximity, and microvascular density, we found that sheathing of subpial vessels by astrocyte processes was continuous along all capillaries, arterioles, and veins, comprising a highly interconnected pathway through which signals could feasibly be relayed over long distances via gap junctions. An inner SR101-positive sheath noted along pial and diving arterioles was determined to be nonastrocytic, and appears to represent selective SR101 staining of arterial endothelial cells. Our findings underscore the intimate relationship between astrocytes and all cortical blood vessels, and suggest that astrocytes could influence neurovascular regulation at a range of sites, including the capillary bed and pial arterioles.

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Scavenging Free Iron Reduces Arteriolar Microvasospasms After Experimental Subarachnoid Hemorrhage

Stroke ◽

10.1161/strokeaha.120.033472 ◽

2021 ◽

Author(s):

Hanhan Liu ◽

Julian Schwarting ◽

Nicole Angela Terpolilli ◽

Kathrin Nehrkorn ◽

Nikolaus Plesnila

Keyword(s):

Subarachnoid Hemorrhage ◽

Cerebral Ischemia ◽

Delayed Cerebral Ischemia ◽

Neurological Deficits ◽

Free Iron ◽

Acute Cerebral Ischemia ◽

Experimental Subarachnoid Hemorrhage ◽

Underlying Mechanisms ◽

Novel Therapeutic Strategies

Background and Purpose: Subarachnoid hemorrhage (SAH) is associated with acute and delayed cerebral ischemia resulting in high acute mortality and severe chronic neurological deficits. Spasms of the pial and intraparenchymal microcirculation (microvasospasms) contribute to acute cerebral ischemia after SAH; however, the underlying mechanisms remain unknown. We hypothesize that free iron (Fe 3+ ) released from hemolytic red blood cells into the subarachnoid space may be involved in microvasospasms formation. Methods: Male C57BL/6 mice (n=8/group) received 200 mg/kg of the iron scavenger deferoxamine or vehicle intravenously and were then subjected to SAH by filament perforation. Microvasospasms of pial and intraparenchymal vessels were imaged three hours after SAH by in vivo 2-photon microscopy. Results: Microvasospasms occurred in all investigated vessel categories down to the capillary level. Deferoxamine significantly reduced the number of microvasospasms after experimental SAH. The effect was almost exclusively observed in larger pial arterioles (>30 µm) covered with blood. Conclusions: These results provide proof-of-principle evidence that Fe 3+ is involved in the formation of arteriolar microvasospasms after SAH and that arteriolar and capillary microvasospasms are triggered by different mechanisms. Deciphering the mechanisms of Fe 3+ -induced microvasospasms may result in novel therapeutic strategies for SAH patients.

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