Lila Khennouf PhD defense – University of Copenhagen

05 September 2017

Lila Khennouf PhD defense

Lila Khennouf  will defend her PhD thesis “Neuronal, glial and pericyte regulation of the brain microcirculation during cortical spreading depression”.

Friday September 8th 15.00 at Medicinsk Museion Auditorium (Bredgade 62, 1260 København K).

Supervisor
Professor Martin Lauritzen, University of Copenhagen

Chair person
Dr Nanna MacAulay, Center for Neurosciences, University of Copenhagen

Opponents
Prof Messoud Ashin (chairman), Dept of Clinical Medicine, University of Copenhagen
Dr Jens Dreier, Charité, Center for Stroke Research, Berlin, Germany
Assoc Prof Cenk Ayata, Harvard University, Boston, USA

ABSTRACT

Cortical spreading depression (CSD) is a depolarization wave that propagates across the grey matter, depolarizing neurons and glial cells. In its mild form it causes the aura in migraine patients, but it can lead to neuronal death in energy-compromised tissue. Clinical and experimental evidences suggest that CSD is involved not only in migraines, but also in the mechanism of stroke, subarachnoid hemorrhage and traumatic brain injury. Many metabolic changes occur during CSD, including changes in cerebral blood flow (CBF), tissue oxygenation and cellular oxygen consumption. The vascular changes during CSD are characterized by a small and short lasting constriction, which is followed by a large vasodilation and a long lasting constriction. Simultaneously, CSD causes a drop in the cortical tissue oxygen down to hypoxic values. After the CSD, the brain metabolic responses to stimulation, i.e. the neurovascular coupling, are reduced. The mechanisms underlying this impairment and the metabolic changes are still unknown, and their understanding could facilitate therapeutic solutions for patient suffering from a CSD. 

In the first paper presented in this thesis, I studied neurovascular coupling and CSD in a mouse model of familial hemiplegic migraine type 1 (FHM1). FHM1 is an autosomal dominant subtype of migraine with a severe prolonged aura of visual and sensory symptoms. The disease is caused by a gain-offunction mutation on the CaV2.1 (P/Q-type) calcium channels, which causes an increased susceptibility to CSD. The mechanisms underlying the disease, the CSD and the prolonged aura remained incompletely understood. To study these mechanisms, I investigate the CBF, oxygen tissue tension, oxygen consumption of the cortex during CSD and during somatosensory stimulations in vivo in wild type (WT) mice and FHM1 mice. In addition I also measured the calcium signal from neurons and astrocytes during CSD and during somatosensory stimulations. CSD resulted in a reduced CBF that took longer time to return to baseline in FHM1 mice compared to WT. The decrease in tissue oxygen during CSD was more severe for FHM1 mice compared to WT, and even dropped to anoxic levels. The measurements also revealed that the oxygen consumed during CSD was higher in FHM1 mice, and was reflected by increased elevation of cytosolic calcium in neurons and astrocytes for FHM1 compared to WT. The neurovascular coupling is the relation between the brain activity and the regulation of cerebral blood flow. Before CSD, responses in CBF and cytosolic calcium to the stimulation were lower in FHM1 mice compared to WT, reflecting an alteration of the neurovascular coupling. CSD caused overall reductions in neurovascular coupling responses to somatosensory stimulation, but CBF and calcium responses were more severely impaired in FHM1 mice. Taken together, these findings suggest that the larger calcium increases, the higher oxygen consumption and the anoxia observed in FHM1 mice compared to WT mice during CSD reduce the metabolic responses to somatosensory stimulation in FHM1 mice. Both the acute and persistent consequences of CSD in FHM1 mice might give rise to the prolonged, severe nature of the somatosensory and visual migraine aura in patients with FHM1. 

The second paper of this thesis gives a detailed assessment of the vasculature changes during CSD, in relation to the variations in pericytes activity. Pericytes are specialized vascular cells covering brain capillaries extensively and they belong to the neurovascular unit. They are essential for normal brain function, such as modulation of the microcirculation or preservation of the blood-brain-barrier during development, but they can also be involved in neuropathologies. Pericytes have been shown to induce capillary constriction after cerebral ischemia, thus, they could potentially be involved in the long lasting constriction observed during CSD. This project explored whether constriction of cortical capillaries by pericytes may contribute to the persistent decrease in CBF and the impaired neurovascular coupling after CSD. To do so, I compared the diameter changes of penetrating arterioles and 1st to 3rd order capillaries in the cerebral cortex of mice in vivo, during CSD and somatosensory stimulation. To compare the activity in capillary pericytes and vascular smooth muscle that surround penetrating arterioles, I engineered a genetically encoded calcium indicator. Both pericytes and vascular smooth muscle cells were successfully transfected, allowing the measurement of cytosolic calcium in these cells. The results showed that the large dilation observed during CSD is mainly driven by penetrating arterioles, while the following long lasting constriction is largest at 1st order capillaries. This strong and long lasting constriction of capillaries was coupled with a large increase in pericyte calcium, indicating their active role in blood flow regulation during CSD. During somatosensory stimulation, penetrating arterioles and capillaries dilated, and this was coupled to a decrease in cytosolic calcium for vascular smooth muscle cells and pericytes. After the CSD, the dilation was impaired for all vessel types, and the calcium decrease in the smooth muscle cells and pericytes was smaller than before CSD. Prevention of pericyte calcium rises during CSD may reduce the persistent blood flow decrease that accompanies migraine and acutely injured human brain cortex. 

In conclusion, I showed in this thesis that CSD evokes changes not only on the oxygen and cerebral blood flow, but also on the calcium activity of neurons, astrocytes, vascular smooth muscle cells and pericytes. The neurovascular coupling used to be defined as the neuronal control over the brain microcirculation, but the results of this thesis emphasize that astrocytes and pericytes are also active component of the neurovascular unit. Further research on their physiology and on how they control the microcirculation will not only bring knowledge to normal brain function, but will also help understanding the mechanism of several neuropathologies.