Repeated longitudinal in vivo imaging of neuro-glio-vascular unit at the peripheral boundary of ischemia in mouse cerebral cortex.

Understanding the cellular events evoked at the peripheral boundary of cerebral ischemia is critical for therapeutic outcome against the insult of cerebral ischemia. The present study reports a repeated longitudinal imaging for cellular-scale changes of neuro-glia-vascular unit at the boundary of cerebral ischemia in mouse cerebral cortex in vivo. Two-photon microscopy was used to trace the longitudinal changes of cortical microvasculature and astroglia following permanent middle cerebral artery occlusion (MCAO). We found that sulforhodamine 101 (SR101), a previously-known marker of astroglia, provide a bright signal in the vessels soon after the intraperitoneal injection, and that intensity was sufficient to detect the microvasculature up to a depth of 0.8 mm. After 5-8 h from the injection of SR101, cortical astroglia was also imaged up to a depth of 0.4 mm. After 1 day from MCAO, some microvessels showed a closure of the lumen space in the occluded MCA territory, leading to a restructuring of microvascular networks up to 7 days after MCAO. At the regions of the distorted microvasculature, an increase in the number of cells labeled with SR101 was detected, which was found as due to labeled neurons. Immunohistochemical results further showed that ischemia provokes neuronal uptake of SR101, which delineate a boundary between dying and surviving cells at the peripheral zone of ischemia in vivo. Finally, reproducibility of the MCAO model was evaluated with magnetic resonance imaging (MRI) in a different animal group, which showed the consistent infarct volume at the MCA territory over the subjects.

Physiological role of brain endothelin in the central autonomic control: from neuron to knockout mouse.

Although endothelin (ET) was discovered as a potent vascular endothelium-derived constricting peptide, its presumed physiological and pathophysiological roles are now considered much more diverse than originally though. Endothelin in the brain is thought to be deeply involved in the central autonomic control and consequent cardiorespiratory homeostasis, possibly as a neuromodulator or a hormone that functions locally in an autocrine/paracrine manner or widely through delivery by the cerebrospinal fluid (CSF). This notion is based on the following lines of evidence. (1) Mature ET, its precursors, converting enzymes, and receptors all are detected at strategic sites in the central nervous system (CNS), especially those controlling the autonomic functions. (2) The ET is present in the CSF at concentrations higher than in the plasma. (3) There is a topographical correspondence of ET and its receptors in the CNS. (4) The ET is released by primary cultures of hypothalamic neurons. (5) When ET binds to its receptors, intracellular calcium channels. (6) An intracerebroventricular or topical application of ET to CNS sites elicits a pattern of cardiorespiratory changes accompanied by responses of vasomotor and respiratory neurons. (7) Recently generated knockout mice with disrupted genes encoding ET-1 exhibited, along with malformations in a subset of the tissues of neural crest cell lineage, cardiorespiratory abnormalities including elevation of arterial pressure, sympathetic overactivity, and impairment of the respiratory reflex. Definitive evidence is expected from thorough analyses of knockout mice by applying conventional experimental methods.

Action of endothelin-1 on vasomotor neurons in rat rostral ventrolateral medulla.

Intracisternal administration of endothelin-1 (ET-1) elicits sympathetically mediated cardiovascular responses by acting on the ventral surface of the medulla oblongata (VSM) subjacent to the rostral ventrolateral medulla (RVLM). We examined, in urethane-anesthetized rats, whether intracisternal ET-1 affected activity of vasomotor neurons (VMNs) in the RVLM, by acting either directly on the VMNs or indirectly via the VSM. VMNs were identified electrophysiologically. Intracisternal administration of ET-1 altered activity of all the 13 VMNs tested. At a dose of 0.1 pmol, ET-1 invariably caused transient excitation in six VMNs examined, whereas at a dose of 1 pmol in separate experiments all the seven VMNs tested were inhibited with (n = 6) or without (n = 1) preceding excitation. Similarly, topical application of ET-1 (0.1-1 pmol) to the VSM caused inhibition with (n = 3) or without (n = 2) preceding excitation in all the five VMNs tested. Direct iontophoretic application of ET-1 to the VMNs caused excitation in four of seven VMNs examined but did not affect the other three neurons. These results support the view that intracisternally administered ET-1 alters activity of VMNs in the RVLM, by acting directly on neurons themselves and indirectly via the VSM.

Endothelin-sensitive areas in the ventral surface of the rat medulla.

In urethane-anesthetized rats, subregions of the ventral surface of the medulla (VSM) in which endothelin (ET) caused cardiorespiratory effects were mapped by topically applying 1 pmol of ET-1. Two distinct subregions, termed the rostral and caudal ET-sensitive areas, were identified. The rostral area was also sensitive to L-glutamate and glycine. It extended between the caudal end of the trapezoid body and the rootlet of the XIIth nerve partly overlying the pyramidal tract. In this position ET-1 caused the type I response consisting of an initial increase (excitatory component) in arterial pressure (AP), renal sympathetic nerve activity (RSNA), heart rate (HR), phrenic nerve activity (PNA) and the number of bursts of PNA (burst rate) followed by a sustained decrease (inhibitory component) in them. The caudal ET-sensitive area was located near the rootlet of the XIIth nerve. In this position ET-1 caused the type II response consisting of a decrease in PNA and an increase in burst rate. Part of this area responded to nicotine but not to glutamate or glycine. ET-3 (10 pmol) applied to the two ET-sensitive areas produced responses similar to those elicited by ET-1. The dose-response relationship was investigated by delivering ETs to the rostral area. The excitatory component of most of the variables was elicited at a dose of 1 fmol of ET-1 or 1 pmol of ET-3, whereas the inhibitory component was produced at 10 fmol of ET-1 or 10 pmol of ET-3. These results suggest that subregions of the rat's VSM may participate in the central cardiorespiratory control by ET.