Involvement of Endothelial Nitric Oxide Synthase in Cerebral Microcirculation and Oxygenation in Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) leads to cerebral microvascular dysfunction and cerebral ischemia. Endothelial nitric oxide synthase (eNOS) is a key regulator of vascular homeostasis. We aimed to assess the role of eNOS in cerebral blood flow (CBF) changes after TBI. Moderate TBI was induced in eNOS knockout (KO) and wild-type (WT) mice (8 per group). Cerebral microvascular tone, microvascular CBF (mCBF) and tissue oxygenation (NADH) were measured by 2-photon laser scanning microscopy (2PLSM) before and 1 hr, 1 and 3 days after TBI. Cerebrovascular reactivity (CVR) was evaluated by the hypercapnia test. Laser Doppler cortical flux (cLDF) was simultaneously measured in the perilesional area. One hr after TBI, cLDF was 59.4 ± 8.2% and 60.3 ± 9.1% from the baseline (p<0.05) in WT and eNOS, respectively. 2PLSM showed decreased arteriolar diameter, the # of functioning capillaries, mCBF, and tissue oxygenation (p<0.05). At 1 day, cLDF increased to 65.2 ± 6.4% in the WT group, while decreased to 56.1 ± 7.2% in the eNOS mice. 2PLSM revealed a further decrease in the # of functioning capillaries, mCBF, and oxygen supply which was slightly milder in WT mice (p<0.05 from the baseline). On the third day after TBI, cLDF increased to 72 ± 5.2% in the WT, while stayed the same in the eNOS group (55.9 ± 6.4%, p<0.05 from the WT). 2PLSM showed reduction in arterioles with vasospasm, increase in the # of functioning capillaries, and improvement in mCBF, and tissue oxygen supply in WT, while no significant changes were observed in eNOS KO (p<0.05). CVR was impaired in both groups 1 hr after TBI, and improved by the 3rd day in the WT, while stayed impaired in eNOS KO. In the subacute TBI period, the significance of eNOS in maintaining cerebral microcirculation and oxygen supply increases with time after the injury.

1. Introduction

Post-traumatic cerebral microvascular dysfunction, characterized by vasoconstriction and vascular occlusion, ultimately leads to cerebral ischemia following traumatic brain injury (TBI) [1]. Nitric oxide (NO), produced by vascular endothelial nitric oxide synthase (eNOS), is a central regulator of vascular homeostasis and cerebral blood flow [2]. Several studies showed that eNOS is essential for the maintenance of cerebral blood flow after TBI [3-5]. However, the involvement of eNOS and NO in TBI pathophysiology is still underinvestigated. The objective of this pilot work was to assess the involvement of eNOS in cerebral microvascular changes after TBI by comparing it in eNOS knockout (KO) and wild-type (WT) mice. Our working hypothesis was that eNOS mediates cerebral microvascular and oxygen supply changes after TBI in a time-dependent manner.

2. Materials and Methods

Animal procedures were approved by the Institutional Animal Care and Use Committee of the Lovelace Biomedical Research Institute under the Animal Protocol #FY20-058. Moderate TBI was induced in 8 C57BL/6 wild type and 8 eNOS^−/− knockout mice [6] by Benchmark Controlled Cortical Stereotaxic Impactor (Leica Biosystems, Germany) through craniotomy (5 mm diam, centered at 3.5 mm posterior and left-lateral to bregma) using a 3-mm flat-tip impounder deployed at a velocity of 5 m/sec and depth of 2.0 mm from the cortical surface, as in our previous study [7]. Regional cerebral blood flow (rCBF) was visualized by RFLSI II Laser Speckle Contrast Imaging System (RWD Life Science Co., Shenzhen, China) before and 1 hour, 1 day and 3 days after the TBI. Cortical microvascular tone, microvascular cerebral blood flow (mCBF) and tissue oxygen supply (NADH) were measured by two-photon laser scanning microscopy (2PLSM) before and 1 hour, 1 day and 3 days after the TBI.

Two-Photon Laser Scanning Microscopy.

The number of perfused capillaries, microcirculation, and tissue oxygen supply were visualized in anesthetized mice (2% isoflurane in 70% N₂O and 30% O₂) using Olympus BX 51WI upright microscope and water-immersion XLUMPlan FI 20x/0.95W objective as previously described [7]. Excitation was provided by a Prairie View Ultima multiphoton laser scan unit powered by a Millennia Prime 10 W diode laser source pumping a Tsunami Ti: sapphire laser (Spectra-Physics, Mountain View, CA). Red blood cell flow velocity was measured in microvessels ranging from 3-50 μm diameter up to 500 μm below the surface of the parietal cortex and ear skin. NADH autofluorescence measurement was used to evaluate mitochondrial activity (metabolic status) and tissue oxygenation [8]. In offline analyses using NIH ImageJ software, three-dimensional anatomy of the vasculature in areas of interest was reconstructed from two-dimensional (planar) scans of the fluorescence intensity obtained at successive focal depths in the cortex (XYZ stack).

Cerebrovascular reactivity testing by hypercapnia challenge.

Cerebrovascular reactivity (CVR) was evaluated by measuring changes in arteriolar diameters and NADH (tissue oxygen supply) during hypercapnia test as previously described [7]. Transient hypercapnia was induced by a 60-second increase in CO₂ concentration to 10% in the inhalation mixture through the face mask. Each trial consisted of 3 minutes of baseline data acquisition, followed by 1 minute of the hypercapnia challenge, and 3 minutes of the post-hypercapnic surveillance period.

Statistical analyses

were done using GraphPad Prism software (La Jolla, CA, USA by independent Student’s t-test or Kolmogorov–Smirnov tests where appropriate. Differences between groups and time were determined using a two-way repeated measures ANOVA analysis for multiple comparisons and post hoc testing using the Mann-Whitney U test. Variables are expressed as mean ± SEM. The level of significance was set at 0.05.

3. Results

At a baseline, regional CBF, arteriolar diameter, and capillary flow velocity were slightly less in eNOS KO but not significantly different than in wild-type mice (Fig 1 A, C). However, the number of functional capillaries and tissue oxygen supply (NADH) was similar in both WT and eNOS KO mice (Fig. 1 B, D). In contrast, the baseline hypercapnia test showed that in eNOS KO mice, cerebral arterioles dilated by 30 ± 8% during inhalation of 10% CO2, which was significantly less than in WT mice (45 ± 6%, Fig. 2A). Simultaneous NADH autofluorescence imaging did not show any transient changes in tissue oxygen supply during hypercapnia in WT animals as a result of arteriolar dilatation and increased RBC traffic, reflecting intact mCBF regulation. In eNOS KO mice, hypercapnia caused a slight increase in NADH autofluorescence, corresponding to the impaired CVR (Fig. 2B). However, this NADH change was not statistically different from the WT mice.

Fig. 1.

After traumatic brain injury: (A) regional cerebral blood flow in the peri-lesion cortex dropped in eNOS knockout (KO) mice more significantly than in wild-type (WT); (B) the number of functional capillaries in the peri-lesion cortex was less in eNOS KO mice comparing to WT; (C) the capillary flow velocity in the peri-lesion cortex was less in eNOS KO mice comparing to WT; and (D) tissue oxygen supply (inversely reflected by nicotinamide adenine dinucleotide level, NADH) in the peri-lesion cortex was less in eNOS KO mice comparing to WT. N= 8 per group, * p < 0.05, mean ± SEM.

Fig. 2.

Nitric oxide-dependent cerebrovascular reactivity in wild-type (WT) and eNOS knockout (KO) mice. (A and B) Cerebrovascular reactivity is impaired in eNOS KO mice reflected by less change in the arterioles' diameter during hypercapnia test compared to WT; and (C and D) Nicotinamide adenine dinucleotide (NADH) autofluorescence dynamics during the hypercapnia test. N= 8 per group, * p < 0.05, mean ± SEM.

One hour after the TBI, rCBF fell to 59.4 ± 8.2% and 60.3 ± 9.1% from the baseline in the ipsilateral hemisphere in WT and eNOS mice, respectively (Fig. 1A, p < 0.05) without difference between groups. In the peri-lesion cortex, 2PLSM showed decreased arteriolar diameter, the number of functioning capillaries, mCBF, and tissue oxygen supply without significant difference between the groups (Fig. 1B-D, p < 0.05 from the baseline).

At one day after the TBI, rCBF in the ipsilateral hemisphere increased to 65.2 ± 6.4% in the WT group, while further decreased to 56.1 ± 7.2% in the eNOS mice (Fig. 1 A, p < 0.05). At this time, 2PLSM over the peri-lesion cortex revealed a further decrease in the number of functioning capillaries, mCBF, and tissue oxygen supply which was slightly milder in WT mice than in eNOS KO (Fig. 1 B-D, p < 0.01).

On the third day after TBI, cLDF in the ipsilateral hemisphere further increased to 72 ± 5.2% in the WT group, while stayed the same in the eNOS group (55.9 ± 6.4%, p < 0.05 from the WT, Fig. 1 A). 2PLSM showed reduction in arterioles with vasospasm, increase in the number of functioning capillaries, and improvement in mCBF, and tissue oxygen supply in WT mice, while no significant changes were observed in eNOS knockouts (Fig. 1 B-D, p < 0.05).

CVR was impaired in both groups one hour after TBI (29.3 ± 7.1 and 15.2 ± 5.4% in WT and eNOS KO mice, respectively), and improved by the third day after TBI in the WT group (35.1 ± 6.2%), while remained impaired in eNOS knockout mice (16.2 ± 5.4%, Fig. 2 A, B, p < 0.05). Simultaneous NADH autofluorescence imaging revealed that one hour after TBI, tissue oxygen supply during hypercapnia test transiently decreased by 5.5 ± 0.7% and 6.6 ± 0.6% in WT and eNOS KO mice, respectively, corresponding with impaired CVR. At three days after the TBI, it improved in WT mice (2.5 ± 0.4%) comparing to eNOS KO mice (6.1 ± 0.5%, Fig 2 C, D p < 0.05).

4. Discussion

The differences in cerebral hemodynamic and metabolism between the eNOS KO and the wild-type mice suggest a critical role of the eNOS in preserving cerebral blood flow metabolism in the contused brain after traumatic injury. The obtained results are in agreement with works performed by Haltky et al., who also demonstrated regional CBF reduction in the acute period of TBI [3]. Lundblad et al. did not find a difference in CBF between WT and eNOS KO mice at 3 hours after the TBI, but at 24 hours, CBF was lower in eNOS KO than in WT mice in our studies [4]. However, in contrast to our research, they did not find any differences in the number of perfused capillaries [4].

5. Conclusions

The significance of eNOS in maintaining cerebral microcirculation and tissue oxygen supply increases with time in the subacute period of TBI. The impaired vasoreactivity due to limited NO production in eNOS KO mice is responsible for poor outcomes. eNOS knockout mouse transgenic model in combination with NOS inhibitors and NO donors might be helpful for the works investigating NO-targeted TBI therapies.