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Despite the recent description of meningeal lymphatic vessels draining solutes from the brain interstitium and cerebrospinal fluid (CSF), the physiological factors governing cranial lymphatic efflux remain largely unexplored. In agreement with recent findings, cervical lymphatic drainage of 70 kD and 2000 kD fluorescent tracers injected into the adult mouse cortex was significantly impaired in the anesthetized compared to waking animals (tracer distribution across 2.1 ± 4.5% and 23.7 ± 15.8% of deep cervical lymph nodes, respectively); however, free-breathing anesthetized mice were markedly hypercapnic and acidemic (paCO2 = 64 ± 8 mmHg; pH = 7.22 ± 0.05). Mechanical ventilation normalized arterial blood gases in anesthetized animals, and rescued lymphatic efflux of interstitial solutes in anesthetized mice. Experimental hypercapnia blocked cervical lymphatic efflux of intraparenchymal tracers. When tracers were injected into the subarachnoid CSF compartment, glymphatic influx into brain tissue was virtually abolished by hypercapnia, while lymphatic drainage was not appreciably altered. These findings demonstrate that cervical lymphatic drainage of interstitial solutes is, in part, regulated by upstream changes in glymphatic CSF-interstitial fluid exchange. Further, they suggest that maintaining physiological blood gas values in studies of glymphatic exchange and meningeal lymphatic drainage may be critical to defining the physiological regulation of these processes.

Local blood flow in the brain is tightly coupled to metabolic demands, a phenomenon termed functional hyperemia. Both capillaries and arterioles contribute to the hyperemic response to neuronal activity via different mechanisms and timescales. The nature and specific signaling involved in the hyperemic response of capillaries versus arterioles, and their temporal relationship are not fully defined. We determined the time-dependent changes in capillary flux and diameter versus arteriolar velocity and flow following whisker stimulation using optical microangiography (OMAG) and two-photon microscopy. We further characterized depth-resolved responses of individual capillaries versus capillary networks. We hypothesized that capillaries respond first to neuronal activation, and that they exhibit a coordinated response mediated via endothelial-derived epoxyeicosatrienoates (EETs) acting on pericytes. To visualize peri-capillary pericytes, we used Tie2-GFP/NG2-DsRed mice, and to determine the role of endothelial-derived EETs, we compared cerebrovascular responses to whisker stimulation between wild-type mice and mice with lower endothelial EETs (Tie2-hsEH). We found that capillaries respond immediately to neuronal activation in an orchestrated network-level manner, a response attenuated in Tie2-hsEH and inhibited by blocking EETs action on pericytes. These results demonstrate that capillaries are first responders during functional hyperemia, and that they exhibit a network-level response mediated via endothelial-derived EETs’ action on peri-capillary pericytes.

Cerebral edema is a major contributor to morbidity associated with traumatic brain injury (TBI). The methods involved in most rodent models of TBI, including head fixation, opening of the skull, and prolonged anesthesia, likely alter TBI development and reduce secondary injury. We report the development of a closed-skull model of murine TBI, which minimizes time of anesthesia, allows the monitoring of intracranial pressure (ICP), and can be modulated to produce mild and moderate grade TBI. In this model, we characterized changes in aquaporin-4 (AQP4) expression and localization after mild and moderate TBI. We found that global AQP4 expression after TBI was generally increased; however, analysis of AQP4 localization revealed that the most prominent effect of TBI on AQP4 was the loss of polarized localization at endfoot processes of reactive astrocytes. This AQP4 dysregulation peaked at 7 days after injury and was largely indistinguishable between mild and moderate grade TBI for the first 2 weeks after injury. Within the same model, blood–brain barrieranalysis of variance permeability, cerebral edema, and ICP largely normalized within 7 days after moderate TBI. These findings suggest that changes in AQP4 expression and localization may not contribute to cerebral edema formation, but rather may represent a compensatory mechanism to facilitate its resolution.

Glial calcium (Ca2+) waves constitute a means to spread signals between glial cells and to neighboring neurons and blood vessels. These waves occur spontaneously in Bergmann glia (BG) of the mouse cerebellar cortex in vivo. Here, we tested three hypotheses: (1) aging and reduced blood oxygen saturation alters wave activity; (2) glial Ca2+ waves change cerebral oxygen metabolism; and (3) neuronal and glial wave activity is correlated. We used two-photon microscopy in the cerebellar cortexes of adult (8- to 15-week-old) and aging (48- to 80-week-old) ketamine-anesthetized mice after bolus loading with OGB-1/AM and SR101. We report that the occurrence of spontaneous waves is 20 times more frequent in the cerebellar cortex of aging as compared with adult mice, which correlated with a reduction in resting brain oxygen tension. In adult mice, spontaneous glial wave activity increased on reducing resting brain oxygen tension, and ATP-evoked glial waves reduced the tissue O2 tension. Finally, although spontaneous Purkinje cell (PC) activity was not associated with increased glia wave activity, spontaneous glial waves did affect intracellular Ca2+ activity in PCs. The increased wave activity during aging, as well as low resting brain oxygen tension, suggests a relationship between glial waves, brain energy homeostasis, and pathology.

Pial arterioles supplying the hindlimb somatosensory cortex dilate in response to electrical stimulation of the contralateral sciatic nerve. The mechanism of this response is not well understood. One possibility involves the cell-to-cell conduction of dilator signals via gap junctions from parenchymal to pial vessels. To test the hypothesis that sciatic nerve stimulation (SNS)-induced dilation of pial arterioles involves gap junctional communication, we determined if pharmacological gap junction blockers may prevent the dilation response. We performed a craniotomy over the somatosensory cortex of adult chloralose-anesthetized rat to create an open cranial window. A surface electrode was placed in the same area for somatosensory evoked potential (SEP) recording. Through the craniotomy, a segment of a pial arteriole overlying the hindlimb somatosensory cortex was chosen for study (Figure 1). Microscopic streams of the gap junction blockers octanol (4 mM) and carbenoxolone (75 microM) were applied continuously onto a segment of the chosen pial arteriole by pressure-ejection using micropipettes, while vessel diameter was monitored at sites upstream and downstream from the site of application relative to the direction of blood flow (Fig. 1). Warmed mock cerebrospinal fluid was continuously superfused to disperse drugs applied by micropipette. At the upstream site, octanol (n=6) and carbenoxolone (n=6) reversibly attenuated SNS-induced dilation to 35% and 30% of the baseline response, respectively. The gap junction blockers did not alter the dilation response at the downstream site. Moreover, the blockade was not due to nonspecific impairment of the response by octanol and carbenoxolone, because 1) dilation to hypercapnia remained intact at both upsteam and downstream sites, and 2) SEP did not change. In addition, we evaluated the effect of connexin mimetic peptides homologous to extracellular loop motifs of vascular connexins, on SNS-induced pial dilation. The gap junction peptides, Gap27(37,43) [SRPTEKTIFII, 250 microM, n=5] and Gap26(37,40) [VCYDQAFPISHIR, 250microM, n=5], were superfused over the pial surface for 120 minutes while the dilation response to SNS was observed at 15-minute intervals. Both peptides significantly reduced SNS-induced dilation (by 70% and 50%, respectively), without affecting SEP. These results suggest that the dilation response of pial arterioles to somatosensory stimulation involves the conduction of dilator signals via gap junctions along the vascular wall.