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. 2021 Jun;41(6):1313-1327.
doi: 10.1177/0271678X20962594. Epub 2020 Oct 13.

Impaired capillary-to-arteriolar electrical signaling after traumatic brain injury

Amreen Mughal  1 , Adrian M Sackheim  2 , Maria Sancho  1 , Thomas A Longden  3 , Sheila Russell  2 , Warren Lockette  4 , Mark T Nelson  1   5 , Kalev Freeman  1   2
Affiliations

Impaired capillary-to-arteriolar electrical signaling after traumatic brain injury

Amreen Mughal et al. J Cereb Blood Flow Metab. 2021 Jun.

Abstract

Traumatic brain injury (TBI) acutely impairs dynamic regulation of local cerebral blood flow, but long-term (>72 h) effects on functional hyperemia are unknown. Functional hyperemia depends on capillary endothelial cell inward rectifier potassium channels (Kir2.1) responding to potassium (K+) released during neuronal activity to produce a regenerative, hyperpolarizing electrical signal that propagates from capillaries to dilate upstream penetrating arterioles. We hypothesized that TBI causes widespread disruption of electrical signaling from capillaries-to-arterioles through impairment of Kir2.1 channel function. We randomized mice to TBI or control groups and allowed them to recover for 4 to 7 days post-injury. We measured in vivo cerebral hemodynamics and arteriolar responses to local stimulation of capillaries with 10 mM K+ using multiphoton laser scanning microscopy through a cranial window under urethane and α-chloralose anesthesia. Capillary angio-architecture was not significantly affected following injury. However, K+-induced hyperemia was significantly impaired. Electrophysiology recordings in freshly isolated capillary endothelial cells revealed diminished Ba2+-sensitive Kir2.1 currents, consistent with a reduction in channel function. In pressurized cerebral arteries isolated from TBI mice, K+ failed to elicit the vasodilation seen in controls. We conclude that disruption of endothelial Kir2.1 channel function impairs capillary-to-arteriole electrical signaling, contributing to altered cerebral hemodynamics after TBI.

Keywords: Cerebral blood flow; capillary endothelial cells; functional hyperemia; inward rectifier K+ channels (Kir2.1); traumatic brain injury.

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Conflict of interest statement

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Cortical capillary structure is preserved after TBI. (a) In vivo 3D-images processed by autotube showing cortical capillaries from a control (i) and TBI (ii) mouse. Block width = 425 × 425 µm, block depth = 300 µm. Summary data comparing various control and TBI structural analyses. No significant differences were present in (b) the number of branch points (166 ± 28, n = 5 vs 201 ± 19, n = 4 branch points, n.s., unpaired t test) (c) vessel width (7 ± 0.4, n = 5 vs 6 ± 1 µm, n = 4, n.s., unpaired t test), or (d) vessel length (8 ± 1, n = 5 vs 9 ± 1 µm, n = 4, n.s., unpaired t test) between control and TBI mice, respectively. Data are expressed as mean ± SD.
Figure 2.
Figure 2.
K+-induced hyperemia is impaired in vivo in TBI mice. (a) In vivo imaging approach. A cranial window was prepared over the somatosensory cortex and imaged using 2-photon laser-scanning microscopy. (b and c) Left: baseline and peak distance–time plots of capillary line scans showing hyperemia to the ejection of 10 mM K+ onto a capillary. RBCs passing through the line-scanned capillary appear as black shadows against green fluorescent plasma. Middle: typical experimental time-course of RBC flux binned at 1-s intervals before and after pressure ejection of 10 mM K+ (300 ms, 5 ± 1 psi; gray arrow) onto a capillary, demonstrating hyperemia to K+ delivery. Right: summary RBC flux responses to 10 mM K+ in (b) control and (c) TBI mice. K+ delivery caused significant hyperemia in control (16 ± 8 vs 31 ± 10 cells/s, n = 8 paired experiments, 6 mice; *p = 0.0004, paired t test), but not in TBI (24 ± 15 vs 25 ± 11 cells/s, n = 8 paired experiments, 6 mice; p = 0.819, paired t test) mice when compared to their baseline prior to K+ application, respectively. (d) The percent change in RBC flux after 10 mM K+ application is significantly decreased in TBI mice when compared to controls (0.6 ± 7 vs 15 ± 7%, n = 8; *p = 0.0008, Mann-Whitney test). (e) K+-induced hyperemia caused a significant increase in flux velocity in control (left) (441 ± 179 vs 840 ± 353 µm/s, n = 8 paired experiments, 6 mice; *p = 0.0022, paired t test) but not in (right) TBI (594 ± 195 vs 726 ± 289 µm/s, n = 8 paired experiments, 6 mice; n.s., paired t test) mice when compared to their respective baseline controls. Data are expressed as mean ± SD.
Figure 3.
Figure 3.
K+-induced capillary-to-arteriolar signaling is impaired in vivo after TBI. Micrograph illustrating pipette placement adjacent to a third-order capillary for in vivo monitoring of the diameter of the upstream feed arteriole (boxed) in (‘ai’) control and (‘aii’) TBI mice. Note the dilation in the penetrating arteriole (boxed). Magnification of the boxed areas around the penetrating arteriole, illustrate the magnitude of dilation evoked by capillary stimulation with 10 mM K+. Summary data showing arteriole diameter before and after capillary application of 10 mM K+, which produced significant upstream arteriole dilation when comparing diameters pre- and post-application of 10 mM K+ in (b) control (10 ± 2 vs 13 ± 3 µm, n = 7 paired experiments, 6 mice; *p < 0.0001, paired t test) but not in (c) TBI (10 ± 2 vs 10 ± 3 µm, n = 7 paired experiments, 6 mice; n.s., paired t test) mice. (d) The change in diameter after application of 10 mM K+ is significantly impaired in TBI (0.7 ± 0.6 µm, n = 7, *p < 0.001, unpaired t test) mice when compared to controls (2 ± 0.2 µm, n = 7). Data are expressed as mean ± SD.
Figure 4.
Figure 4.
KIR2.1 channel current density is markedly reduced in ECs isolated from TBI mice. Inwardly rectifying current (black) was evoked by a 400-ms voltage ramp (−140 to +40 mV; lower inset) in capillary endothelial cells isolated from control (a) and TBI (b) mice and blocked by 100 μM Ba2+. (c) Summary data of Ba2+-sensitive current density at peak inward currents (−140 mV) in control (−11 ± 2 pA/pF, n = 8 cells, from 3 mice) and TBI mice (−4 ± 1 pA/pF, n = 11 cells, from 4 mice) (*p < 0.0001, Mann-Whitney test). Data are expressed as mean ± SD.
Figure 5.
Figure 5.
Vascular reactivity is impaired in pial arteries following TBI. Representative traces of lumen diameter in PCAs from (a) control and (b) TBI mice. Extracellular K+ was raised from 3 to 10 mM (highlighted in gray). The vasodilatory response was measured at the point of maximal dilation and normalized to maximal diameter (“passive diameter”), obtained in zero Ca2+ aCSF with 100 µM diltiazem at the end of each experiment. (c) Summary data for peak diameter elicited by 10 mM K+ in PCAs from control (56 ± 32%; n = 7) and TBI (26 ± 16%; n = 7) mice (*p < 0.05; unpaired t test). (d) Summary data for spontaneous myogenic tone at 80 mm Hg in PCAs from control (15 ± 4%; n = 7) and TBI (23 ± 16%; n = 7) mice (n.s.; unpaired t test). Data are expressed as mean ± SD.
Figure 6.
Figure 6.
Proposed mechanism showing impairment of electrical signaling after TBI. Left panel represents healthy brain where neural activity driven increase in extracellular K+ causes activation of capillary endothelial cell Kir2.1 channels to initiate propagating hyperpolarization (electrical signal) towards upstream penetrating arterioles. At the arteriolar level, these hyperpolarizing signals inhibit voltage gated Ca2+ channels (VDCCs) to relax smooth muscle cells causing dilation of the penetrating arterioles to increase CBF. Right panel represents pathophysiology of TBI on the brain resulting in disruption of capillary-to-arteriolar electrical signaling and impaired K+-driven hyperemic response.
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