STEM I

Taught by Dr. Crowthers

This class gives students the opportunity to create their own five-month long research project. It teaches how to gather information from other previous experiments, summarize research journals, perform elevator pitches, and execute your project idea. There are also mini-projects throughout the year, such as the Build Something project.

My Project

The Effects of Nitric Oxide on the Cerebrovascular System during Exercise

Nitric oxide increases cerebral blood flow (CBF) and cerebral partial oxygen pressure (PO2) during exercise, which may not have been enhanced by ethyl nitrite at 20 ppm. However, a loss in nitric oxide production may have a direct effect on the amount of brain oxygen delivery.

Abstract

During high-intensity exercises, the need for oxygen delivery increases significantly. Cerebral blood flow decreases, showing a lack of oxygen delivery in the cerebral vascular system. As a result, an individual may experience fuzzy vision, drowsy eyes, and blackouts. In order to provide an effective and efficient method for athletes, this project proposes to accelerate and maintain the production of nitric oxide (NO) in the body. Nitric oxide is a molecule that can vasodilate — increasing blood flow and oxygen delivery. This function can be used to determine if NO is a viable option for athletes. Additionally, nitric oxide is a naturally occurring molecule in the human body and it can be produced by many enzymes. In this experiment, mice are used to model the physiological effects for humans. The mice followed three forced exercise programs and were linked up to a cerebral blood flow (CBF) sensor, a partial oxygen pressure (PO2) sensor, and an electrocardiogram (ECG) sensor. When the data was collected and analyzed, the results were compared to previous studies in order to determine the effectiveness of NO. These tests support that nitric oxide production is directly related to change in cerebral blood flow during exercise, and inhaled ethyl nitrite during forced exercise does not significantly increase cerebral blood flow, but this may be due to low exercise intensity. Forced exercise on endothelial nitric oxide synthase (eNOS) — another nitric oxide generator — knockout mice will be conducted in the future to observe the importance of eNOS in the brain. Keywords: Nitric Oxide, cerebral blood flow, cerebral partial oxygen pressure, ethyl nitrite, hypoxia exercise

Graphical Abstract


Research Thesis

Click here to access my supporting documents.



Research Question

Research Hypothesis

How does nitric oxide influence the behavior of cerebral blood flow and partial oxygen pressure during high-intensity exercise?

If nitric oxide (NO) generation increases during mice forced exercise, then NO mediated cerebral blood flow and oxygen delivery will be increased.



Background

During high-intensity exercises, athletes experience fatigue due to sore muscles and a lack of blood flow and oxygen delivery in the brain. This can cause hazy vision, dizziness, and sometimes blackouts. During heavy exercise, the vast increase in cardiac output is directed almost exclusively to contracting skeletal and cardiac muscles (Joyner et al, 2015). This causes blood flow to target less towards the brain, the control center of the body. Exercise-induced muscle damage (EIMD) arises when muscles become damaged after unfamiliar and strenuous exercise. It is a condition characterized by delayed onset muscle soreness, swelling, impaired muscle function, and increased inflammation (Corr et al., 2021). The symptoms of delayed onset muscle soreness (DOMS) set in about 24 hours after the athlete has completed their exercise (Lewis et al., 2012). There are 6 theories for the mechanism of DOMS: lactic acid accumulation, muscle spasm, microtrauma, connective tissue damage, inflammation, electrolytes, and enzyme efflux. These mechanisms strain the muscle’s functional unit — the sarcomere — such as the contraction during exercise. At the same time, the brain is also pressured to function. The brain function is critically dependent on sufficient oxygen supply by cerebral vasculature because of its high oxygen metabolism rate and lack of oxygen reserve (Moeini et al., 2020). Exercise changes both the cerebral metabolic rate of oxygen and increases the demand on the cardiovascular system (Rooks et al, 2010; Duncker et al, 2008). Therefore, oxygen demand in the skeletal muscles distracts blood flow by directing it towards supplying the skeletal muscles with oxygen rather than the cerebral vasculature.

While performing a high-intensity exercise, the cerebral blood flow can only increase to a certain limit that is controlled by the brain’s autoregulation of blood flow speed (Moeini et al, 2020). Evidence shows that autoregulation tends to lower the cerebral blood flow speed with increasing exercise intensity. A study conducted by Tan et al., 2014, the bolded line depicts the cerebral blood flow across three exercise intensities. As the exercise intensity increases, cerebral blood flow also increases. However, during a point between moderate and heavy intensity, the cerebral blood flow starts to decrease. This behavior is most likely due to the brain’s autoregulation, which is a point of capacity where the volume of cerebral blood flow can not surpass (Moeini et al., 2020).

There is a period where cerebral blood flow decreases while the exercise intensity increases. This may cause symptoms such as haziness in vision, dizziness, shortness of breath, and sometimes even blackouts (Aráujo, 2021; Mayo Clinic, 2020; Ziegelstein, 2004). Blackouts can cause neurological problems such as memory loss, confusion, and physical injuries during the blackout episode. There are many methods to prevent blackouts. One of the major interests is nitric oxide (NO), which exists in the human body. NO is a key factor that maintains cardiovascular homeostasis and promotes relaxation through the cyclic guanosine monophosphate (cGMP) pathway, a pathway that is found mainly in the skeletal muscles (Ma et al., 2021; Kobzik et al., 1994). These behaviors can greatly increase the delivery of oxygen in cerebral blood flow and the endurance of an athlete.

However, during high-intensity exercise, NO produced by existing NO generators in the body cannot vasodilate for long periods, hence, additional NO producers are needed. A common practice in supplying additional NO is the inhalation of NO, but this practice generates toxic components harmful to the body, but other methods can be applied that can stimulate proteins to generate more NO and cause no harm to the user (Moya et al., 2002). Two of the many methods are increasing NO synthase (NOS) and ethyl nitrite (ENO) in the vascular system in order to improve NO effects, which are crucial in maintaining homeostasis during exercise. Inhaled ENO enters the bloodstream through the alveoli and stimulates through nitric oxide synthase (NOS) which increases NO production. ENO is safe to inhale and causes minimal harm to the body of the user (Moya et al., 2002).

In addition to ENO, another nitric oxide generator is NOS, the primary producer of nitric oxide in the vascular system. NOS has three types: endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS). As stated in the name, eNOS is found in the endothelium, and it produces the most nitric oxide molecules when compared with nNos and iNOS. A reduced availability of eNOS has been shown to significantly slow the recovery time of cerebral blood cells after ischemic stroke (Zhu et al., 2016). eNOS plays a significant role in maintaining cerebral homeostasis by preserving CBF, inhibiting inflammation and apoptosis (Zhu et al., 2016).

Another protein that produces NO is S-nitrosohemoglobin. This protein obtains the ability to transport and release NO throughout the body because of the cysteinebeta93 amino acids (C93) (Stamler et al., 1997). Hemoglobin proteins exist in two forms: R structure (high O2 affinity) and T structure (low O2 affinity). Through activation of NO in C93, the hemoglobin protein becomes S-nitrosohemoglobin. This activation commonly occurs during the T structure since there is a low PO2 concentration in the vascular system. In a study conducted in 1997 by Jonathan Stamler et al., it was found that S-nitrosohemoglobin increases the blood flow in the brain and vascular system. If the C93 amino acid is transformed into alanine in a mouse (C93A), then the hemoglobin protein loses the ability to intake NO, which reduces S-nitrosohemoglobin in the vascular system.

BackgroundInfographic


Procedure

Cys93A Mutant Mice Experiment Prep

A mouse (Cys93A mutant or WT) was placed on the treadmill with its head fixed to the device that eliminates head movement during exercise. The CBF sensor was then connected to a Laser Doppler, and the ECG sensors were connected to a bio amplifier. The mouse then followed a running test. First, its baseline CBF was recorded. Second, the mouse ran for 5 minutes on the treadmill at 3 m/min and its average CBF was recorded. Third, it rested for 5 minutes or until the mouse’s CBF returned to baseline. Fourth, the mouse repeated the same exercise and the average CBF was recorded. Finally, the mouse rested until its CBF returned to baseline. It was then taken off the treadmill and placed in its cage with food and water. This experiment test was performed for all mice that were assigned to this experiment.

Inhaled-air vs. Inhaled-ENO in Normoxia Experiment (WT Mice) Prep

A WT mouse was placed on the treadmill with its head fixed to the device that eliminates head movement during exercise. The CBF sensor and PO2 sensor was the connected to a Laser Doppler, and the ECG sensors were connected to a bio amplifier. The mouse then performed a running test. First, its baseline CBF and PO2 while inhaling air were recorded. Second, the mouse ran for 5 minutes on the treadmill at 3 m/min and its average CBF and PO2 were recorded. Third, it rested for 5 minutes for its CBF and PO2 to return to baseline. Fourth, the air inhaled-trial was switched to an inhaled-ENO trial (inhaling ENO at 20 ppm while running on the treadmill for 5 minutes at 3m/min) and the average baseline CBF and PO2 were recorded. Fifth, the same exercise that was performed in the air trial was repeated in the ENO-trial, and the average CBF and PO2 were recorded. Finally, the mouse rested until its CBF returned to baseline. It was then taken off the treadmill and placed in its cage with food and water. This experiment test was performed for all mice that were assigned to this experiment.

Inhaled-air vs. Inhaled-ENO in Hypoxia Experiment (WT Mice) Prep

The equipment setup and mouse genotype are the same as the experiment test in 2.5. The test trials are similar to the test trials in 2.5: the first test trial is inhaled-air trial in normoxia, the second is inhaled-air trial in hypoxia (10% O2), the third is inhaled-ENO trial in hypoxia (10% O2).

eNOS Knockout Mice Experiment Prep

The equipment setup of this experiment is the same as the C93A mutant mice experiment. Additionally, this experiment only measured CBF. Instead of using C93A mutant mice, this experiment utilizes three young eNOS knockout (KO) mutant mice. The test trials of this experiment follow the same exercise test as the C93A mutant mice experiment.

ExperimentTests


Figures

Figure 1

Figure 2

CBf Normoxia Data PO2 Normoxia Data

Figure 3

Figure 4

CBF Hypoxia Data PO2 Hypoxia Data

Figure 5

Figure 6

CBF eNOS Data PO2 eNOS Data

Figure 7

Figure 8

Change in CBF eNOS Data Change in PO2 eNOS Data


Data Analysis

Note* Due to a low sample size, a significance/confidence value cannot be determined. Instead, the trends of the graphs will be analyzed.

From Figure 1, it is observed that the increase in CBF of the WT mice during exercise is larger than the increase in CBF of the C93A mutant mice. This shows that a reduced ability to transport nitric oxide may decrease the change of CBF during exercise. Following this trend, it is expected that C93A mutant mice have a lower increase in CBF when experiencing strenuous exercise. Therefore, nitric oxide transportability may be directly related to CBF response during exercise.

In Figure 2 and Figure 3, the amount of CBF and PO2 during the resting periods of the inhaling-air trial and inhaling-ENO trial are respectively similar. This observation is also seen in the treadmill periods of the trials. From this data, ENO may not have effect on the increase of CBF and PO2 during exercise. This is unexpected since ENO has been shown to increase blood flow is the pulmonary system and cardiovascular system (Moya et al., 2002). A possible factor of these results may be a low sample size, meaning that data is not enough to observe a trend, or it may be due a low-exercise intensity, where it does not need excess nitric oxide to increase its CBF and PO2. Due to the results of this experiment, it influence the next experiment: the same variables as the current experiment, but the mice performed it in a hypoxia (10% O2) environment.

In Figure 4 and Figure 5, inhaling ENO in a hypoxia environment did not increase the amount of CBF and PO2 during exercise. In all figures, it is observed that the amount of CBF and PO2 during the treadmill periods are the lowest out of the three trials. If the trend continues, then the hypoxia-ENO trial will have the lowest CBF and PO2 for all periods. From these figures, ENO may not have a significant contribution to the increase of CBF and oxygen delivery during exercise. Similar to the previous experiment, these results may be due a low sample size or low-exercise intensity. Due to the heads of the mice being fixed in one position, the mice cannot run at a strenuous pace. A possible alternative is prolonging the exercise.

In Figure 6 and Figure 7, the eNOS knockout (KO) mice had an overall lower CBF than the WT mice, but an overall higher PO2. If more samples were collected, it is expected to see the CBF of the eNOS KO mice during resting and treadmill periods to be lower than the WT mice. From this data, a reduced production of nitric oxide may decrease the amount of CBF. The amount of PO2 from the eNOS KO mice is unexpected as it is more than the WT mice. This infers that a reduced production of nitric oxide may increase the amount of cerebral PO2. From Figure 8, the change in CBF of the eNOS KO mice may be similar to the change in CBF of the WT mice, however, more samples are needed to be collected. From Figure 9, the change in PO2 of the eNOS KO mice may be less than the change in PO2 of the WT mice, which is expected since there is a decreased production of nitric oxide.



Discussion and Conclusion

Discussion

Since the sample size right now is small, a significance test can not be performed. Instead, trends of current data and predictions for future data are explained.

The Cys93A mutant mice experiment showed that the lack of Cys93 results in a low change in cerebral blood flow. Since Cys93 is responsible for S-nitrosylation, the vascular system of the mutant mice lacks the S-nitrosohemoglobin protein, therefore, the mice lack an efficient method of transporting NO throughout the body. In this experiment, the change in CBF of the WT mice was about 1500 BPU, while the change in CBF of the C93A mutant mice was about 300 BPU. It is clearly shown the mutant mice could not increase their CBF, which may be due to the lack of C93 amino acids in the hemoglobin. Though the sample size is small, a trend can be seen: the change in CBF of WT mice during exercise may be significantly greater than that of the mutant mice.

The inhaled-air vs. inhaled-ENO in normoxia experiment did not show major differences between inhaling air and inhaling ENO during exercise. The amount of CBF and PO2 for both the inhaling-air trial and inhaling-ENO trial were relatively the same during the rest periods and treadmill periods. With more samples, a difference might occur, but following the observed trend, inhaling ENO does not have a significant effect on the cerebrovascular system during exercise. This may be due to the low exercise intensity, but the speed of the treadmill can not increase due to the head of the mice being fixed in a position. If the exercise intensity is not high enough for the brain to require an abundance of oxygen, then the use of nitric oxide in blood vessels may be limited. Therefore, there is no need for excess nitric oxide during this experiment, so the actual effects of ENO may not be shown. The inhaled-air vs. inhaled-ENO in hypoxia experiment is based off of this prediction. In hypoxia, oxygen intake is limited, so the mice should theoretically require more oxygen in their cerebrovascular system when exercising.

The inhaled-air vs. inhaled-ENO in hypoxia experiment showed differences between the normoxia inhaled-air trial (inhaled-air), hypoxia inhaled-air trial (inhaled-10%O2), and hypoxia inhaled-ENO (inhaled-10%O2 ENO) trial. The CBF of the inhaled-air trial and inhaled-10%O2 trial during the rest and treadmill periods were relatively similar, but there was an observable difference between CBF of these two trials and the inhaled-10%O2 ENO trial. Additionally, the percent change of CBF in the inhaled-air trial was observably greater than the percent change of CBF in the hypoxia trials. This CBF data shows that ENO may not help increase the amount of CBF, and it may not contribute to boosting the change in CBF. This may be due to a low exercise intensity, but more samples are needed to be collected. The PO2 of the hypoxia trials are observed to be relatively similar, and the PO2 of the inhaled-air trial is greater than the hypoxia trials. Additionally, the percent change of PO2 in the hypoxia trials were observed to be greater than the percent change of PO2 in the inhaled-air trial. This data shows the amount of PO2 in a hypoxia environment is less than the amount of PO2 in normoxia. However, the percent change of PO2 in hypoxia is greater than in normoxia. This shows that there may be a sensitive PO2 response to exercise in a low-oxygen concentrated environment. ENO may not have an effect on the high percent increase of PO2 because the inhaled-10%O2 trial and inhaled-10%O2 ENO trial have relatively the same amount of PO2 and percent change of PO2. Following these trends, ENO will most likely have little effect on the percent increase of PO2, and it will most likely not have an effect on the amount of CBF and the percent increase of CBF. A larger sample size is needed, and the exercise intensity also needs to be increased further in order to reveal the effects of ENO.

In Figure 6 and Figure 7, the eNOS knockout (KO) mice had an overall lower CBF than the WT mice, but an overall higher PO2. If more samples were collected, it is expected to see the CBF of the eNOS KO mice during resting and treadmill periods to be lower than the WT mice. From this data, a reduced production of nitric oxide may decrease the amount of CBF. The amount of PO2 from the eNOS KO mice is unexpected as it is more than the WT mice. This infers that a reduced production of nitric oxide may increase the amount of cerebral PO2. From Figure 8, the change in CBF of the eNOS KO mice may be similar to the change in CBF of the WT mice, however, more samples are needed to be collected. From Figure 9, the change in PO2 of the eNOS KO mice may be less than the change in PO2 of the WT mice, which is expected since there is a decreased production of nitric oxide.

Conclusion

Further tests need to be conducted for each experiment in order to achieve an accurate significance value. According to the trends in the CBF graph of the WT mice and C93A mutant mice, NO may have an impact on the increase of CBF during exercise, and the test methods do increase the CBF of the mice, so these methods can be incorporated into other experiments. According to the trends in the inhaled-air vs. inhaled-ENO graphs, ENO may not obtain the ability to increase CBF and PO2 during exercise, which may be due to the low exercise intensity during the treadmill periods. From the eNOS knockout mice experiment graphs, a reduced production of NO from eNOS can be a viable method to further the research of the effects of NO on CBF and brain PO2. The trends of CBF and PO2 in the eNOS knockout mice may be similar to those observed in the C93A mutant mice experiment.



References

Project Poster