Mild Intermittent Hypoxia and CPAP: A Multi-pronged Approach to Treat Sleep Apnea in Intact and Spinal Cord Injured Humans
Phase: NA
Conditions: Obstructive Sleep Apnea, Spinal Cord Injuries
Sponsor: Wayne State University

 
 
 
 
 
 
 
 
 
Mild Intermittent Hypoxia and CPAP: A Multi-Pronged Approach to Treat Sleep 
Apnea in Intact and Spinal Cord Injured Humans 
 
Record # 1 R56HL142757-01 
Date: November 5th 2018 
 
 

Experimental Protocol 
Specific Aim 1. To determine if IH coupled with CPAP mitigates autonomic, cardiovascular, 
neurocognitive and metabolic measures both directly and indirectly by improving CPAP 
compliance as a consequence of LTF of upper airway muscle activity initiated by exposure to IH. 
a. Rationale. Many studies have reported that IH directly mitigates co‐morbidities typically 
associated with OSA. In addition, exposure to IH may increase CPAP compliance by initiating 
LTF of upper airway muscle activity that could enhance upper airway patency leading to a 
reduction in therapeutic CPAP. Reduced pressures could result in improved compliance and 
outcome measure. 
b. Recruitment. The participants with OSA may be recruited from Sleep Disorders Centers 
affiliated with Wayne State University and John D. Dingell Veterans Administration Medical 
Center. The total number of patients studied in these laboratories per year is approximately 4500 
and about 40 % of the participants will meet the inclusion criteria. We will also target hypertension 
clinics at John D. Dingell VA Medical Center and the Detroit Medical Center as additional sites to 
recruit participants with coincident hypertension and OSA; since up to 30 % of individuals 
diagnosed with hypertension also have been diagnosed with OSA. Advertisements will also be 
placed in the local newspaper (i.e. Metro Times) and on various websites (i.e. Craigslist, 
Facebook and Wayne State University Pipeline). During recently completed studies 
approximately 100 individuals were screened in order to recruit 20 participants that met the 
inclusion criteria. In the present proposal we believe this rate of recruitment will improve because 
our inclusion criteria is less stringent than in the past (i.e. previously we recruited individuals with 
OSA that fell within a narrow age range and did not suffer from other co‐morbid conditions). Based 
on past experience, once participants meet the inclusion criteria and have successfully completed 
the screening sleep study and practice trial few drop out beyond this point. Nonetheless we have 
taken into account that 2 participants from each group (see Participants below) may drop out 
beyond the point of the screening and practice trials, so an additional 4 participants may be 
enrolled if required. 
c. Participants. To complete the R56 proposal, Hypoxia group will be comprised of 4 participants 
with OSA that will be treated with IH and CPAP. Sham group will be comprised of 4 participants 
with OSA that will be exposed to a sham protocol and CPAP. Participants will be randomized to 
Hypoxia or Sham group. Participants will be male or female of any race, 18‐60 years of age with 
a BMI of less than 40 kg/m2 and pure or predominantly (i.e. comprised of both a central and 
obstructive component) OSA (AHI ≤ 80 events per hour and an average oxygen desaturation level 
of 85 % or greater). Participants will not have received CPAP treatment previously. Participants’ 
will be diagnosed with Stage 1 to Stage 2 hypertension as categorized by the 2018 American 
Heart Association guidelines. Systolic and diastolic pressure will range from 130 ‐ 159 mmHg and 
80 ‐ 99 mmHg, respectively. Participants with a systolic pressure greater than 159 mmHg or a 
diastolic pressure greater than 99 mmHg will be excluded from the study. Based on the 
characteristics of this group the short exposure to mild IH that we will induce does not pose a risk 
beyond what exists for participants without OSA based on previous studies we have completed. 
Moreover, the nocturnal oxygen desaturation routinely experienced by the recruited participants 
will be mild, which will reduce the possibility of central nervous system hypoxic depression. Thus, 
we believe that initiation of various forms of plasticity following exposure to IH may manifest 
themselves more clearly in these individuals. The participants will be free of any other known 
cardiovascular disease. The participants will not be on any medications used to treat 

cardiovascular disorders or any sleep promoting supplements including melatonin. All participants 
will have normal lung function with no or minimal alcohol consumption (< 2 oz of alcohol/night). 
Females in the Hypoxia and Sham groups will be studied at similar points in their menstrual cycle. 
The participants will not be night shift workers or recently travelled across time zones. These 
criteria will be confirmed by the completion of health, lifestyle, and sleep questionnaires, from 
pulmonary function tests, a physical examination and a nocturnal polysomnography examination 
that will be completed at John D. Dingell Veterans Administration Medical Center. During the 
physical examination blood pressure will be measured and a baseline EKG will be obtained. 
Assessment of cricomental space, pharyngeal grade, overbite, tonsillar grade, palatal position 
and tongue size will be completed. Completing this assessment will allow us to ensure that upper 
airway dimensions are similar between OSA participants that are being treated with CPAP and 
IH, compared to the group that will be only be treated with CPAP. A pregnancy test will be 
completed on female participants to avoid fetal exposure to IH. 
d. Protocol and Measurements. Visit 1: Health, lifestyle and sleep questionnaires (i.e. SF‐36 
Health survey and Epworth Sleepiness Scale) along with pulmonary function tests, blood pressure 
measurements, a 12‐lead electrocardiogram and a physical examination will be completed. Visit 
2: A sleep study beginning at approximately 10 pm will be completed. The study will be used to 
confirm the presence or absence of OSA. Visit 3: A sleep study will be completed to determine 
the therapeutic positive airway pressure required to eliminate flow limitation. In addition, the 
pressure associated with airway collapsibility will be determined (i.e. the critical closing pressure 
‐ PCRIT). Upon awakening participants in Hypoxia group will undergo a trial exposure to IH (two 
2‐minute episodes of 8% oxygen separated by two‐minute normoxic intervals, with an 
accompanied 2 mmHg increase in carbon dioxide levels) while sham group will be exposed to 
room air in order to acclimate to the equipment and accompanying sensations. Visit 4: Following 
the sleep study, participants will be given a blood pressure holter monitor (ABPM‐05, Meditech 
Inc. Framingham, MA) to measure blood pressure and pulse rate at home over a 24‐hour period. 
Data collection will begin at 6 am on a weekend day. Blood pressure values will be obtained every 
20 minutes. Participants will be given a diary and an actigraph watch to document activities 
completed over the 24‐hour period. Thereafter data will be standardized relative to activity level 
(i.e. quiet and active wakefulness and sleep). Participants will be encouraged to adhere to a daily 
routine that will be replicated following completion of the IH protocol. These measures are being 
made to monitor the changes in blood pressure associated with activities of daily living. Visits 5 ‐ 
19: Following the completion of visits 1‐4, participants will visit the lab 15 times over a 3‐week 
period during the day. The participants will visit the laboratory each week day for 3 weeks. If a 
week day visit is missed it will be replaced by a visit on a week end day. During each visit, 
participants in Hypoxia group will be exposed to 12 two‐minute episodes of 8% oxygen combined 
with a 2 mmHg increase in carbon dioxide separated by two minute normoxic intervals. The IH 
protocol will be comprised of a 20‐minute baseline period followed by exposure to twelve ‐ two 
minute episodes of hypoxia [partial pressure of end‐tidal oxygen (PETO2) = 50 mmHg]. Each 
episode will be interspersed with a 2‐minute recovery period under normoxic conditions. The 
PETCO2 will be sustained 2 mmHg above baseline values for the last ten minutes of baseline 
and throughout the remainder of the protocol. In addition to the IH protocol, a group of participants 
will also be exposed to a sham protocol in addition to being treated with CPAP during sleep. The 
sham protocol will be administered during wakefulness for a minimum of 15 days over a 3‐week 
period. During the sham protocol the participants will be exposed to atmospheric levels of oxygen 
and carbon dioxide for the duration of the IH protocol. During the IH and sham protocol respiratory 

(ventilation, PETCO2, PETO2, oxygen saturation) and cardiovascular (heart rate and EKG) 
parameters will be monitored continuously throughout each visit. Likewise, with the exception of 
visits 5, 12 and 19 (see immediately below) automated and auscultatory blood pressure 
measurements will be obtained prior to the onset of the protocol, during hypoxic episodes 1, 6 
and 12, during recovery from these episodes, as well as, at the midpoint and completion of the 
end recovery period of the IH or sham protocol. On each day of the protocol participants will 
continue to be treated with CPAP each night while they sleep in their home environment with the 
exception of visits 12 and 19 (see Table 1 below). They will be instructed to use CPAP each night 
for as long as possible.  
 
 
Visits 5, 12 & 19: On visits 5, 12 & 19 additional autonomic, cardiovascular, neurocognitive and 
metabolic measures will be obtained. Upon entering the laboratory participants will complete a 
series of questionnaires and tasks to assess vigilance and neurocognitive function. The 
Pathfinder Number Test and Psychomotor Vigilance Task will assess attention and psychomotor 
function. The Buschke Selective Reminding Test will assess learning and memory. The Epworth 
Sleepiness Scale will assess day time sleepiness. The Mini Mental State Examination will be used 

to assess five areas of cognition function (i.e. orientation, registration attention, calculation, recall 
and language). Thereafter, 15 ml of blood will be sampled prior to exposure to the IH or sham 
protocol to identify potential metabolic (i.e. lipid profile, hemoglobin A1C), inflammatory (i.e. 
asymmetric dimethylarginine and high sensitivity C‐reactive protein) and angiogenic/vasculogenic 
(hypoxia inducible factor 1α and vascular endothelial growth factor) biomarkers that might reflect 
the efficacy of exposure to IH. Thereafter, on visits 5 & 19 beat to beat measures of blood pressure 
will be obtained throughout the IH or sham protocol. Beat to beat measures of blood pressure will 
also be obtained on visit 12. On visits 12 & 19 daytime exposure to the IH or sham protocol will 
be followed by a sleep study completed in the laboratory on the evening following the day time 
visit. Measures obtained during these sleep studies will be similar to the measures described for 
Visit 3. Visit 20: The protocol outlined for Visit 4 will be repeated on Visit 20. 
Data and Statistical Analysis. 
Research staff that will not administer the gases will be assigned to complete a blind analysis of 
all measures. The staff assigned to analyze data (e.g. ventilation and blood pressure) collected 
during the direct application of hypoxia might be able to determine the applied stimulus based on 
the respiratory or cardiovascular response. However, the criteria used to analyze the data is 
rigorous and bias is unlikely. The Shaprio‐Wilk test will be used to test if the data is normally 
distributed. Assuming that the data will be normally distributed, a two‐way analysis of variance 
with repeated measures in conjunction with Student‐Newman Keuls post‐hoc test will be used to 
determine if differences in 24 hour blood pressure measures are evident before and after 
exposure to IH or the sham protocol. The two factors in the design will “Group (Hypoxia vs. sham)” 
and “time point (Visit 4 vs. Visit 20).” A similar analysis will be used to determine if primary (i.e. 
blood pressure, autonomic nervous system activity, therapeutic CPAP, critical closing pressure, 
limb motor function tests) and secondary (vigilance and neurocognitive measures and blood 
biomarkers) outcome measures differ before, midway and after completion of the IH or sham 
protocol. The two factors in the design will be “Group (Hypoxia vs. sham)” and “time point (Visit 5 
vs. Visit 12 vs. Visit 19)”. Primary outcome variables collected during IH or the sham protocol (i.e. 
blood pressure, autonomic nervous system activity, minute ventilation, tidal volume, breathing 
frequency, PETCO2, PETO2) will be analyzed using a three‐way analysis of variance with 
repeated measures in conjunction with Student‐Newman Keuls post hoc. The three factors in the 
design will be “Group (Hypoxia vs. sham)”, “Visit Number (Visit 5 vs. Visit 12 vs. Visit 19)” and 
“Time Point (Baseline vs. Initial hypoxic episode vs. Final hypoxic episode vs recovery of IH or 
sham protocol).” A Pearson product‐moment correlation and regression analysis will be used to 
examine the relationship between CPAP adherence (e.g. percentage of days CPAP used over 4 
or 6 hours) during the last half of the protocol with changes in the primary and secondary outcome 
measures for hypoxia and sham groups. If CPAP adherence impacts on the primary and/or 
secondary outcome measures we expect the slope of the relationship will be greater in Hypoxia 
group compared to sham group (i.e. for a given level of adherence modification to the primary or 
secondary outcome variable will be greater in the CPAP and IH group compared to the CPAP 
and sham group). 
Potential Problems & Solutions. (i) The use of a parallel design could increase the burden of 
recruiting participants. However, we have extensive experience in recruiting and retaining 
participants enrolled in parallel design protocols requiring multiple visits. This statement is further 
supported by the participants that completed the protocol during collection of our preliminary data. 
Use of a cross‐over design would make blinding of the participants difficult and would extend the 

IH protocol from 3 to at least 10 weeks, since a wash‐out period would be required between IH 
and sham. Given concerns that the protocol could be too long, we believe a parallel design is 
appropriate. (ii) Exposure to mild IH during sleep in untreated participants might result in 
increased breathing instability. This potential problem will not be an issue in the present study. 
Participants will be exposed to IH while awake during daytime hours. Likewise, participants will 
be treated with CPAP during sleep and we anticipate that therapeutic CPAP will be reduced while 
sleep apnea is effectively treated as the protocol progresses. Nonetheless, the CPAP instrument 
that we utilize monitors the AHI each night. If there is an unexpected increase in apnea severity 
the participant will be asked to withdraw from the study. (iii) We anticipate that sympathetic 
nervous system activity and blood pressure will be reduced in response to mild forms of IH 
coupled with CPAP. However, if progressive increases in blood pressure are evident over 3 visits 
that equate to sustained increases in systolic and diastolic blood pressure of greater than 10 
mmHg the participants will be withdrawn from the study. Because the participants are coming to 
the laboratory each day changes in health status will be monitored day to day. (iv) The 
instrumentation and methods employed during sleep on Visits 3, 5, 12 and 19 could lead to 
disruption of sleep and the inability to collect the planned measures. Given the population we are 
employing this is typically not an issue when the participants are treated with CPAP. Nonetheless, 
all measurements will be obtained during deep N2 or N3 of non‐rapid eye movement sleep and a 
sleep efficiency of at least 75 % will be required. If these criteria are not obtained a repeat sleep 
study will be completed on the following day. (v) Participants will be blinded to the composition of 
the gas mixture. However, they may recognize increases in heart rate and breathing during IH 
exposure. During the consenting process we will describe potential responses that might be 
experienced during each visit to all participants, without linking the responses to IH exposure. 
Based on past experience naïve participants often do not directly associate increases in breathing 
and heart rate with IH. Nevertheless, at least one of our outcome measures will allow us to 
differentiate if knowledge of the gas mixture, or other factors, influenced our results. More 
specifically, measures of the critical closing pressure during sleep should not be influenced by 
knowledge of the gas mixture. If CPAP compliance improves without a coincident improvement 
in the critical closing pressure we would question if knowledge of the gas mixture, or other factors, 
are influencing our results.