Impact of NOS, COX, and ROS Inhibition on Cerebral Blood Flow Regulation

Specific Aims:

1A. Determine the independent contributions of NOS and COX to hypoxic and hypercapnic
vasodilation in the MCA of young, healthy adults.

1B. Determine the combined contribution of NOS and COX to hypoxic and hypercapnic
vasodilation in the MCA of young, healthy adults.

1. C. Determine the combined contribution of NOS, COX, and ROS to hypoxic and hypercapnic
vasodilation in the MCA of young, healthy adults.

2. A. Determine the independent contributions of NOS and COX to hypoxic and hypercapnic
vasodilation in the BA of young, healthy adults.

2B. Determine the combined contribution of NOS and COX to hypoxic and hypercapnic
vasodilation in the BA of young, healthy adults.

2C. Determine the combined contribution of NOS, COX, and ROS to hypoxic and hypercapnic
vasodilation in the BA of young, healthy adults.

Initial Screening: A phone screening questionnaire will determine whether or not potential
subjects meet preliminary eligibility criteria. Potentially eligible subjects will be invited
for an additional in-person screening. Subjects will need to come to the screening visit
having fasted (except water) for a minimum of 10 hours.

Laboratory screening procedures include:

1. Informed consent

2. Health history questionnaire

3. Physical activity questionnaire

4. Urine pregnancy test (females only)

5. Venous blood draw

6. DEXA scan (dual energy x-ray absorptiometry)

Study Design: Eligible subjects will complete 2 study visits examining hypoxic and
hypercapnic responses under four conditions: control, NOS inhibition or COX inhibition, NOS
and COX inhibition and NOS, COX, and ROS inhibition. During visit 1, subjects will receive
L-NMMA (NG-Monomethyl-L-arginine; NOS inhibition), ketorolac (COX inhibition), then ascorbic
acid (ROS inhibition). During visit 2, subjects will receive ketorolac, L-NMMA, then ascorbic
acid. Study visits will be conducted in a randomized, counterbalanced order and all drugs
will be infused intravenously. Hypoxia will elicit an SPO2~80% as determined by pulse
oximetry and hypercapnia will increase end-tidal CO2 (PETCO2) by ~10 mmHg from baseline.
Throughout each visit, subjects will be monitored for heart rate, blood pressure, pulse
oximetry oxygen saturation, respiratory gases, ventilation, and CBF velocity. After
instrumentation and baseline data collection, hypoxic and hypercapnic trials will commence
with middle cerebral artery velocity (MCAv), basilar artery velocity (BAv), respiratory, and
cardiovascular variables being recorded. All trials will be separated by 10-minutes of quiet
rest while breathing room air and will not be randomized due to the order and timing of drug
infusions required to explore our aims. Due to the difficulty of insonating the BA, this will
serve as an exploratory aim and not deemed a necessary outcome for study completion.

Experimental Methods and Intervention Plan:

Transcranial Doppler Ultrasound (TCD) MCAv will be measured via the transtemporal window
while BAv will be measured through the transforaminal window with 2-MHz (megahertz)
transcranial Doppler ultrasound probes. In approximately 30% of individuals the BA cannot be
identified; therefore, insonation of the MCA alone will be deemed sufficient to proceed with
the study in cases in which BA cannot be located.

Measurements Height and weight will be measured to calculate body mass index (BMI). Waist and
hip circumferences will be measured as indicators of regional adiposity. DEXA scan will be
used for determination of body composition. Venous blood samples will be obtained for the
determination of blood chemistry values. During each visit, subjects will be studied in a
semi-recumbent position and instrumented for continuous measurement of heart rate (3-lead
ECG), pulse oximetry oxygen saturation (SPO2, pulse oximeter), and blood pressure (MABP,
automated physiological monitor). Hemodynamic parameters will be continuously measured with
finger plethysmography. Inspiratory and expiratory gases will be measured with a gas analyzer
and respiratory flow will be determined with a heated pneumotachometer.

Hypoxia Hypoxia will be used to cause cerebral vasodilation under resting, semi-recumbent
conditions. Three isocapnic hypoxia trials will be performed per study visit. Subjects will
inspire through a two-way non-rebreathing valve, connected to a gas mixer, supplied by
medical grade pressurized oxygen (O2), carbon dioxide (CO2), and nitrogen (N2). After
3-minutes of baseline room air breathing, hypoxia will be introduced by decreasing inspired
O2 (~11% O2) to elicit and sustain 3-minutes of SPO2~80% as determined by a pulse oximeter.
After 3-minutes of baseline steady state hypoxia, there will be a 5-minute drug loading
period (L-NMMA, ketoroloc, or ascorbic acid), followed by a 5-minute drug maintenance period.
Hypoxia will be maintained during this time, with total hypoxia duration of ~15-minutes per
each hypoxia trial. Isocapnia will be achieved through the addition of CO2 to inspired gas.
End-tidal CO2 (PETCO2) has been shown to be a valid predictor of arterial blood CO2 levels.
Prior data from our lab indicate that steady state hypoxia is achieved within 3 minutes.
Additionally, hypoxia eliciting an SPO2~80% will be examined as cerebral vessels have
relatively low cerebrovascular sensitivity to less severe hypoxia (SPO2~90%). A total of 3
hypoxia trials will be conducted per study visit yielding ~45 minutes of steady state
hypoxia.

Hypercapnia An increase in systemic CO2 is a powerful signal to increase blood flow within
the cerebral circulation. A total of 4 hypercapnic trials will be performed during each study
visit. Subjects will inspire through a three-way sliding rebreathing valve attached to a
latex balloon containing a hyperoxic (O2=40%), hypercapnic (CO2=3%) gas mixture with the
balance N2. The balloon will be filled to a volume exceeding estimated vital capacity (as
determined by age, sex, and height) by 1-liter. After 3-minutes of baseline room air
breathing, hypercapnia will commence by sliding the three-way rebreathing valve from room air
to the attached latex balloon. During hypercapnia, PETCO2 increases (~10 mmHg above baseline
values), but inspired O2 does not fall below room air percentage of 21%. Hypercapnia will be
sustained (~2-minutes) until PETCO2 values reach ~10 mmHg above baseline values, after which
subject will begin breathing room air. PETCO2 will be used as a reliable, non-invasive
measure of arterial CO2. A total of 4 hypercapnic trials will be conducted per study visit
yielding ~ 8 minutes of hypercapnia per study visit.

Intravenous Catheter Trained lab personnel will place two intravenous catheters (one in each
arm) for each study visit. One catheter will be used for the intravenous infusion of study
drugs (L-NMMA, Ketorolac, and Ascorbic Acid). The second catheter will be used to draw
intermittent blood samples at 8-specific time points throughout each study visit to ensure
pharmaceutical efficacy and examine systemic physiologic blood variables of interest. The
catheter used for blood draws will be kept patent with a 0.9% saline drip. If only one IV
catheter can be placed or one fails, the study will proceed with both drug infusion and blood
draws occurring from a single catheter.

Intravenous L-NMMA Infusion Intravenous L-NMMA is the only non-FDA approved drug that will be
used, but is commonly utilized in the research setting. Intravenous infusion of L-NMMA will
be used to inhibit the enzyme NOS which is responsible for the formation of NO. L-NMMA is a
lyophilized powder that is diluted with saline. In the current study, intravenous L-NMMA will
be administered via a 3 mg kg-1 loading dose over 5-minutes (36 mg kg-1 hr-1), followed by a
maintenance dose of 1 mg kg-1 hr-1 for the remainder of the study. Considering a typical 75
kg individual this will result in a loading dose of 225mg and a maintenance dose (~112min) of
140mg totaling 365 mg L-NMMA throughout the duration of the study, when L-NMMA is the first
study drug infused (L-NMMA infused during HX1, HX2, HX3 and HC2, HC3, HC4). During the visit
when L-NMMA is second study drug infused (L-NMMA infused during HX2, HX3 and HC3, HC4) the
total dosing will be less as the maintenance infusion (~67 min) will be reduced by ~45
minutes totaling 309 mg L-NMMA. The loading dose of L-NMMA is equivalent to that used in a
prior study investigating the contribution of NOS to hypoxic cerebral vasodilation while the
maintenance dose and the absolute amount of L-NMMA infused will be less than previously
utilized.

Intravenous Ketorolac Infusion Intravenous ketorolac is FDA approved non-steroidal
anti-inflammatory drug used for the treatment of pain. Ketorolac will be used to
non-specifically inhibit the enzyme cyclooxygenase which is responsible for the formation of
vasoactive prostaglandins (prostacyclin, thromboxane). Inhibition of prostaglandin synthesis
during hypoxia will allow assessment of the contribution of these vasoactive prostaglandins
to hypoxic vasodilation. Common intravenous doses of ketorolac are 15 mg and 30 mg bolus
infusions; however, ketorolac will be dosed relative to body mass. Ketorolac will be
intravenously administered via a 0.3 mg kg-1 loading dose over 5 minutes (3.6 mg kg-1 hr-1)
with a minimum loading dose of 15 mg. This will then be followed by a maintenance dose of
0.03 mg kg-1 hr-1 for the remainder of the study. Considering a typical 75 kg individual this
will result in a loading dose of 22.5 mg and a maintenance dose (~112min) of 4.2 mg totaling
26.7 mg ketorolac throughout the duration of the study, when ketorolac is the first study
drug infused (ketorolac infused during HX1, HX2, HX3 and HC2, HC3, HC4). During the visit
when ketorolac is second study drug infused ketorolac infused during HX2, HX3 and HC3, HC4)
the total dosing will be less as the maintenance infusion (~67 min) will be reduced by ~45
minutes totaling 25 mg of ketorolac. Similar doses of ketorolac have been shown to reduce
whole body prostanoids and reduce perioperative pain. Plasma ketorolac concentrations peak
within ~3 minutes following intravenous administration with a terminal half-life of 5.6
hours.

Intravenous Ascorbic Acid Infusion Intravenous ascorbic acid is FDA-approved. Ascorbic acid
will be intravenously administered to acutely reduce ROS. Ascorbic acid will be administered
via loading dose of 0.035 g kg fat-free mass-1 over 5-minutes (0.42 g kg fat-free mass-1
hr-1). Followed by a maintenance dose of 0.060 g kg fat-free mass-1 hr-1 over the remaining
study time course (~20 minutes) for continued suppression of reactive oxygen species.
Considering a 75 kg individual with a fat-free tissue mass of ~55kg (based upon previous
young, healthy subjects in our lab), subjects will receive ~ 3.025 g of Ascorbic acid during
the course of a study visit.

Plasma Assays

~10 mL of blood will be drawn during the screening visit as a component of study eligibility.
Screening blood samples will be analyzed for fasting venous glucose, creatinine, total
cholesterol, HDL cholesterol, LDL cholesterol, and triglycerides. Venous blood samples will
be drawn at 8 time points during the study protocol via a venous catheter. Each blood draw
will be 10 mL, totaling 80 mL. Blood will be centrifuged with plasma and serum drawn off and
stored at -80ºC. Efficacy of COX inhibition will be determined by the measurement (EIA) of
the circulating COX metabolites 6-keto-prostaglandin F1α (as stable marker of PGI2) and
thromboxane B2 (a stable marker of TXA2). Efficacy of ROS inhibition will be determined by
plasma vitamin C concentrations and oxidized low-density lipoproteins (oxLDL) serving as a
systemic marker of oxidative stress. Acute intravenous Ascorbic acid administration has been
shown to decrease oxLDL in healthy adults. Efficacy of NOS inhibition will be determined by
sum of nitrite and nitrate (NOx), which is considered to be an index of NO production.

Inclusion Criteria:

1. Age: 18 ≤ years ≤ 45

2. Free of disease and otherwise healthy as determined by health history questionnaire

3. Not currently taking medication with the exception of birth control as determined by
health history questionnaire

4. Low to moderate physical activity will be permitted and assessed by a physical
activity questionnaire (≤ 4 hours of physical activity/week)

5. Body mass index (BMI) < 25 kg/m2

6. Resting blood pressure <140/<90 mmHg (lowest of three measures)

7. Resting heart rate <100 bpm

8. Resting pulse oximetry oxygen saturation (SPO2) >95%

9. Fasting venous blood values (average of two measures)

1. Glucose <100 mg/dL

2. Creatinine < 1.5 mg/dL

3. Total cholesterol <200 mg/dL

i. HDL cholesterol >40 mg/dL (men) ii. HDL cholesterol >50 mg/dL (women) iii. LDL
cholesterol < 130 mg/dL d. Triglycerides <150 mg/dL

10. Subjects must be willing to report to the laboratory on all study days after
completing

1. Minimum 10-hour fast

2. Minimum 18-hours abstention from exercise, alcohol, caffeine, and non-steroidal
anti-inflammatory drugs (i.e. aspirin, ibuprofen, and naproxen)

11. Additionally, women will

1. Have a regular menstrual cycle (self-report)

2. Be studied (study visit 1 and study visit 2) on days 1-5 of menstrual cycle
(self- report).

Exclusion Criteria:

1. Coronary artery disease

2. Stroke

3. Heart attack

4. Heart valve disease

5. Congestive heart failure

6. Previous heart surgery

7. Lung disease

8. Peripheral vascular disease

9. Gastrointestinal (GI) bleeding

10. Allergy or Intolerance to Aspirin or NSAIDS

11. History of renal/kidney disease, insufficiency, or injury

12. Smoke or use tobacco within the last year

13. Subject has an abnormality or contraindication to study participation, which is not
covered in the eligibility criteria.

Additionally, women will be excluded if they are

1. Pregnant (as determined by a urine pregnancy test on screening and study days)

2. Currently breastfeeding (self-report)

3. Post-menopausal (self-report)