Hyperbaric air mobilizes stem cells in humans; a new perspective on the hormetic dose curve

Abstract

Introduction
Hyperbaric air (HBA) was first used pharmaceutically in 1662 to treat lung disease. Extensive use in Europe and North America followed throughout the 19th century to treat pulmonary and neurological disorders. HBA reached its zenith in the early 20th century when cyanotic, moribund “Spanish flu pandemic” patients turned normal color and regained consciousness within minutes after HBA treatment. Since that time the 78% Nitrogen fraction in HBA has been completely displaced by 100% oxygen to create the modern pharmaceutical hyperbaric oxygen therapy (HBOT), a powerful treatment that is FDA approved for multiple indications. Current belief purports oxygen as the active element mobilizing stem progenitor cells (SPCs) in HBOT, but hyperbaric air, which increases tensions of both oxygen and nitrogen, has been untested until now. In this study we test HBA for SPC mobilization, cytokine and chemokine expression, and complete blood count.

Methods
Ten 34–35-year-old healthy volunteers were exposed to 1.27ATA (4 psig/965 mmHg) room air for 90 min, M-F, for 10 exposures over 2-weeks. Venous blood samples were taken: (1) prior to the first exposure (served as the control for each subject), (2) directly after the first exposure (to measure the acute effect), (3) immediately prior to the ninth exposure (to measure the chronic effect), and (4) 3 days after the completion of tenth/final exposure (to assess durability). SPCs were gated by blinded scientists using Flow Cytometry.

Results
SPCs (CD45dim/CD34⁺/CD133⁻) were mobilized by nearly two-fold following 9 exposures (p = 0.02) increasing to three-fold 72-h post completion of the final (10th) exposure (p = 0.008) confirming durability.

Discussion
This research demonstrates that SPCs are mobilized, and cytokines are modulated by hyperbaric air. HBA likely is a therapeutic treatment. Previously published research using HBA placebos should be re-evaluated to reflect a dose treatment finding rather than finding a placebo effect. Our findings of SPC mobilization by HBA support further investigation into hyperbaric air as a pharmaceutical/therapy.

Full text

Frontiers in Neurology 01 frontiersin.org
Hyperbaric air mobilizes stem cells
in humans; a new perspective on
the hormetic dose curve
KentJ.MacLaughlin
1
*, GregoryP.Barton
2, RudolfK.Braun
1,
JuliaE.MacLaughlin
3, Jacob J.Lamers
1, Matthew D.Marcou
1
and MarloweW.Eldridge
1
1 Department of Pediatrics, University of Wisconsin–Madison, Madison, WI, United States, 2 Department
of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States,
3 Medical Oxygen Outpatient Clinic, The American Center, Madison, WI, United States
Introduction: Hyperbaric air (HBA) was first used pharmaceutically in 1662 to treat
lung disease. Extensive use in Europe and North America followed throughout the
19th century to treat pulmonary and neurological disorders. HBA reached its zenith
in the early 20th century when cyanotic, moribund “Spanish flu pandemic” patients
turned normal color and regained consciousness within minutes after HBA treatment.
Since that time the 78% Nitrogen fraction in HBA has been completely displaced
by 100% oxygen to create the modern pharmaceutical hyperbaric oxygen therapy
(HBOT), a powerful treatment that is FDA approved for multiple indications. Current
belief purports oxygen as the active element mobilizing stem progenitor cells (SPCs) in
HBOT, but hyperbaric air, which increases tensions of both oxygen and nitrogen, has
been untested until now. In this study wetest HBA for SPC mobilization, cytokine and
chemokine expression, and complete blood count.
Methods: Ten 34–35-year-old healthy volunteers were exposed to 1.27ATA (4
psig/965 mmHg) room air for 90 min, M-F, for 10 exposures over 2-weeks. Venous
blood samples were taken: (1) prior to the first exposure (served as the control for
each subject), (2) directly after the first exposure (to measure the acute eect), (3)
immediately prior to the ninth exposure (to measure the chronic eect), and (4) 3 days
after the completion of tenth/final exposure (to assess durability). SPCs were gated by
blinded scientists using Flow Cytometry.
Results: SPCs (CD45dim/CD34+/CD133-) were mobilized by nearly two-fold following
9 exposures (p = 0.02) increasing to three-fold 72-h post completion of the final (10th)
exposure (p = 0.008) confirming durability.
Discussion: This research demonstrates that SPCs are mobilized, and cytokines are
modulated by hyperbaric air. HBA likely is a therapeutic treatment. Previously published
research using HBA placebos should be re-evaluated to reflect a dose treatment
finding rather than finding a placebo eect. Our findings of SPC mobilization by HBA
support further investigation into hyperbaric air as a pharmaceutical/therapy.
KEYWORDS
CD34+, Traumatic Brain Injury, CD133, war veteran self harm, war veterans’
psychological suering, war veterans suicide, hyperbaric air placebo, HBOT
Introduction
Presently, hyperbaric air (HBA) is not thought of as a medication. Its singular approved
medicinal use is for Acute Mountain Sickness. Historically it has been used medicinally, initially
reported by Henshaw in 1662 to treat “aictions of the lungs” (1) and additional reports from
OPEN ACCESS
EDITED BY
George Mychaskiw,
Ochsner LSU Health, UnitedStates
REVIEWED BY
Ingrid Eftedal,
Norwegian University of Science and
Technology, Norway
Shai Efrati,
Tel Aviv University, Israel
*CORRESPONDENCE
Kent J. MacLaughlin
kmaclaughlin@pediatrics.wisc.edu
RECEIVED 23 March 2023
ACCEPTED 11 May 2023
PUBLISHED 20 June 2023
CITATION
MacLaughlin KJ, Barton GP, Braun RK,
MacLaughlin JE, Lamers JJ, Marcou MD and
Eldridge MW (2023) Hyperbaric air mobilizes
stem cells in humans; a new perspective on the
hormetic dose curve.
Front. Neurol. 14:1192793.
doi: 10.3389/fneur.2023.1192793
COPYRIGHT
© 2023 MacLaughlin, Barton, Braun,
MacLaughlin, Lamers, Marcou and Eldridge.
This is an open-access article distributed under
the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or
reproduction in other forums is permitted,
provided the original author(s) and the
copyright owner(s) are credited and that the
original publication in this journal is cited, in
accordance with accepted academic practice.
No use, distribution or reproduction is
permitted which does not comply with these
terms.
TYPE Original Research
PUBLISHED 20 June 2023
DOI 10.3389/fneur.2023.1192793

MacLaughlin et al. 10.3389/fneur.2023.1192793
Frontiers in Neurology 02 frontiersin.org
Europe and America followed (1). In 1857 Simpson published a paper
using HBA to treat lung pathologies including tuberculosis (2).
Interest in hyperbaric air as a medication surged following successful
treatments of “Spanish u” patients by Cunningham in 1918 (3–5).
Unfortunately, Cunningham produced a paucity of papers supporting
his work and when hedied in 1937, interest in hyperbaric air abated.
HBA began to be employed again in the modern era with
controversial results. It was used as a placebo by Collet while
investigating the use of hyperbaric oxygen in Cerebral Palsy (6) and
also as a placebo in brain injury by Miller, Wolf and Cifu (7–9). In
each of these experiments, both the treatment group and the placebo
group improved revealing an apparent placebo eect. e use of
hyperbaric air as a placebo is energetically debated and controversial
because it increases the oxygen tension. In contrast to the placebo
ndings, similar published studies not using the hyperbaric air
placebo, found signicant improvements in brain injury (10–20).
Considering the aforementioned, we asked the question, “is
hyperbaric air an appropriate placebo?”. Wesearched the literature
and found no evidence that hyperbaric air has been tested.
Wedesigned a test of hyperbaric air using a gold standard endpoint of
oxygen therapy, stem cell mobilization.
Our specic aims were (1) to characterize stem cell mobilization
in healthy adults following daily exposures to hyperbaric air, (2)
determine if other biomarkers were modulated, (3) determine if there
were acute changes, and (4) if so, were the changes durable. Based on
previous research done in our lab (21) and calculating a 27% increase
in oxygen partial pressures in the inhaled gases, wehypothesized that
(1) stem cells would bemobilized, (2) biomarkers would bemodulated
(3) there would beacute changes and (4) the stem cell mobilization
would bedurable.
Materials and methods
Design and subjects
is prospective hyperbaric air study was a randomized, single-
blind study conducted at the University of Wisconsin – Madison
Clinical Sciences Center between May 1st, 2021, and August 31st,
2021. is study is approved by the Institutional Review Board of the
University of Wisconsin – Madison. UW IRB ID: 2020-0293-CR001.
All participants provided written informed consent. Healthy adults
were recruited for participation in this study (Table1).
Hyperbaric air exposure
All exposures were at 1.27 ATA (4 psig) of room air for 90 min in
a Gamow style Mountain Sickness Chamber (Hyperbaric Technologies
Inc. Amsterdam, NY, UnitedStates). To minimize circadian cell cycle
variations including acrophase and circannual cycle, all subjects were
diurnally active and all hyperbaric exposures and sample collections
occurred at the same time of day over the contiguous
15-day experiment.
Sample collection
Each peripheral venous blood sample was collected during
the subject’s exposure time slot using either a 21- or 23-gauge BD
Vacutainer Safety-Lok Blood Collection Set (Becton, Dickinson
and Company, Franklin Lakes, NJ, UnitedStates) into a Cyto-
Chex BCT tube (Streck Inc. NE USA), BD Vacutainer Plastic
Blood Collection Tubes with K2EDTA, BD Vacutainer Plastic
Blood Collection Tubes – PST Plasma Separation Tubes, and
Greiner Bio-One K2EDTA GelTubes. All samples were stored
according to manufacturer’s directions. Study protocol in graphic
format is included in Figure1.
Flow cytometry
Fluorescence minus one (FMO) tubes, rainbow bead tubes, and
single antibody tubes were prepared as gating references. Antibodies
were pipetted into ow cytometry tubes according to manufacturer’s
instructions. Antibodies used include CD34 = Brilliant Violet 421
(BioLegend, San Diego, CA, UnitedStates), CD45 = Alexa Fluor 488
(BioLegend, San Diego, CA, UnitedStates), CD133 = PE (Miltenyi
Biotec, North Rhine-Westphalia, Germany) CD31 = Brilliant Violet
605 (BioLegend, San Diego, CA, United States), CD105 = PE-Cy7
(BioLegend, San Diego, CA, United States), Ghost Dye Red
780 = Tonbo Biosciences, San Diego, CA.
Flow cytometry was performed by blinded scientists on a
ermoFisher Attune NxT (Waltham, MA, UnitedStates). Samples
were analyzed by blinded scientists using FlowJo soware (FlowJo,
Ashland, OR, UnitedStates).
Enzyme-linked immunosorbent assay
e Invitrogen ProcartaPlex™ Human Immune Monitoring
Panel 65-Plex (Invitrogen, Waltham, MA, UnitedStates) was used to
assess changes in cytokines, chemokines and growth factors. All tests
were performed by blinded scientists at the University of Wisconsin
Non-Human Primate Research Center (Madison, WI, UnitedStates).
Statistical analysis
e rst blood draw taken prior to the rst exposure served as the
control. To determine whether there was an overall eect across
exposures, weutilized the Friedman test (nonparametric alternative
to one-way ANVOA with repeated measures) and if signicant,
comparisons between all-time points were performed using the
Wilcoxon signed rank test. Signicance level was determined a priori
at the 0.05 level and all tests were two-tailed. Statistical analyses were
calculated using Graph Pad Prism (GraphPad Prism 9.0.0 Soware,
San Diego, CA, UnitedStates).
TABLE1 Anthropometric data of subjects in this hyperbaric air study.
NMean Standard
deviation
Age 10 34.54 years 1.36 years
Height 10 171.75 cm 9.38 cm
Weight 10 81.40 kg 21.05 kg
BMI 10 27.43% 2.8%
Female 5 5 NA

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Frontiers in Neurology 03 frontiersin.org
Results
CD45dim
Increased frequency of CD45dim/CD34+/CD133−
following nominal exposure to hyperbaric room
air
As previously described 10 humans were exposed to 1.27 ATA of
room air 10 times over the course of a 12-day period. Results revealed
a signicant increase in the frequency of CD45
dim
/CD34
+
/CD133
−
stem progenitor cells in venous blood resulting in an approximate
two-fold increase directly prior to the 10th exposure (p = 0.02).
CD45
dim
/CD34
+
/CD133
−
SPCs continued to increase during the 3 days
following the end of exposures and increased to three-fold, 72 h aer
the 10th exposure (p = 0.008); (Figure2A).
Decreased frequency of CD45dim/CD34−/CD133+
after hyperbaric exposure
While CD45dim/CD34+/CD133− SPCs increased, the frequency of
CD45
dim
/CD34
−
/CD133
+
primitive pro-angiogenic stem progenitor
cells signicantly decreased aer exposure to intermittent hyperbaric
air. CD45dim/CD34−/CD133+ decreased by nearly ve-fold prior to the
10th exposure (p = 0.02) and mobilization decreased by six-fold 72 h
aer the 10th exposure (p = 0.01) (Figure2B).
FIGURE1
Experimental protocol.
FIGURE2
Frequency of CD45dim/CD34+/CD133-, CD45dim/CD34-/CD133+ and CD45dim/CD31+/CD105-after intermittent hyperbaric air exposure detected by flow
cytometry. (A) CD34+ and CD133- stem progenitor cells. (B) CD34- and CD133+ stem progenitor cells. (C) CD31+ and CD105- stem progenitor cells.

MacLaughlin et al. 10.3389/fneur.2023.1192793
Frontiers in Neurology 04 frontiersin.org
FIGURE3
Frequency of CD45+/CD34+/CD133-, CD45+/CD34-/CD133+, CD45+/CD31+/CD105-, and macrophage derived chemokine after intermittent hyperbaric
air exposure detected by flow cytometry and ELISA. (A) CD34+ and CD133- stem progenitor cells. (B) CD34- and CD133+ stem progenitor cells.
(C) CD31+ and CD105- stem progenitor cells. (D) Macrophage derived chemokine.
Increased frequency of CD45dim/CD31+/CD105−
after hyperbaric exposure
e frequency of CD45dim/CD31
+
/CD105
-
was also
signicantly increased aer hyperbaric exposure by nearly two-fold
prior to the 10th exposure (p = 0.023) and increased to over
two-fold 3 days aer the 10th exposure (p = 0.016) (Figure2C).
Mobilization of CD45+/CD34+/CD133−
The expression of CD45
+
/CD34
+
/CD133
-
also increased by
nearly 3.5 fold after 9 full exposures (p = 0.012) and increased to
over four fold 72 h following the end of the 10th exposure
(p = 0.002) (Figure3A).
Decreased frequency of CD45+/CD34−/
CD133+
A signicant reduction in the expression of CD45
+
/CD34
−
/
CD133+ was noted resulting in a six-fold reduction prior to the 10th
exposure (p = 0.016) and recovering to 5.4-fold 72 h aer the 10th and
nal exposure (p = 0.016) (Figure3B).

MacLaughlin et al. 10.3389/fneur.2023.1192793
Frontiers in Neurology 05 frontiersin.org
Mobilization of CD45+/CD31+/CD105−
e expression of CD45
+
/CD31
+
/105
-
increased by nearly two-fold
aer 9 full exposures (p = 0.02) and remained at the level through 72 h
following the end of the 10th exposure (p = 0.008) (Figure3C).
Complete results from flow cytometry
Complete results from ow cytometry testing, including
non-signicant ndings are included in tabular form for reference.
(Table2).
Macrophage-derived chemokine (MDC)
expression significantly decreased
Results from the Invitrogen ProcartaPlex™ Human Immune
Monitoring Panel 65-Plex ELISA like analysis showed there was only
one change to report. e expression of Macrophage-derived
Chemokine (MDC) was signicantly lower between the second and
third time points (p = 0.008). All other tests revealed no change
(Figure3D).
Complete blood count with dierential
ere were no signicant changes in CBC at any time points
(Table2).
Discussion
e smallest dose of hyperbaric air that will result in a therapeutic
eect is unknown and strongly debated among scientists and physicians.
Pressures below 1.4 atmospheres absolute of hyperbaric air are accepted
as a placebo but have not previously been tested in humans. Studies using
hyperbaric air as a placebo have resulted in a “placebo” or “participation”
eect (7–9). is placebo eect nding is vigorously disputed because
hyperbaric air signicantly increases the partial pressure of oxygen (and
nitrogen) in the inspired air (22–27). e “placebo eect” ndings have
eectively restricted the use of HBOT for many conditions including
Traumatic Brain Injury in soldiers returning from combat.
In this study weasked the question, will a small dose of hyperbaric
air (1.27 ATA), that is below the accepted 1.4 ATA therapeutic
threshold, mobilize stem cells similar to HBOT? Wehypothesized that
stem cells would bemobilized.
Indeed, stem cells were signicantly mobilized, refuting previous
“placebo eect” ndings. Weintend that the results will provide
needed experimental data to medical societies and journal editors,
and in turn provide guidance to FDA and physicians.
Critical analysis of major findings
Testing for stem cell mobilization using ow cytometry and gated by
blinded scientists, the major nding of this study is that 1.27 ATA
hyperbaric air mobilizes CD34+/CD133- SPCs and CD31+/CD105- stem
cells in humans receiving 10 daily 90-min exposures (Figures 2A,C,
respectively). Mobilization of CD34
+
SPCs has also been observed in
isobaric hyperoxic (21) and in hyperbaric hyperoxic conditions (28, 29).
e mechanism of hyperbaric air SPC mobilization in this
experiment is beyond the scope of this study, but may besimilar to
SPC mobilization found in hyperbaric oxygen therapy, which activates
nitric oxide synthase and plays a prime role in initiating CD34
+
SPC
mobilization (30–33). (CD34 background) CD34
+
adult stem/
progenitor cells are a group of specic cell types that possess the
abilities of self-renewal and multipotent dierentiation (34, 35).
CD34
+
is expressed on hematopoietic and pro-angiogenic stem
progenitor cells and on endothelium (36). Pro-angiogenic stem/
progenitor cells contribute to neovascularization by a process of
homing to ischemic tissue called vasculogenesis, and by budding
endothelium from established blood vessels in a process called
angiogenesis (37, 38). Further research is needed to understand the
homing and function of the CD34
+
SPCs mobilized by intermittent
hyperbaric air exposures.
A second signicant nding of this study, which wereport for the
rst time, is hyperbaric air mobilizes CD31
+
/CD105
-
SPCs. is novel
nding has many implications in the eld of hyperbaric air and
hyperbaric oxygen. (CD31
+
background) CD31 is thought to have a
protective role in experimental atherosclerosis (39). e therapeutic
potential of CD31 agonists to manage atherosclerotic disease
manifestations is a consistent nding in pre-clinical studies (40).
Although this is the rst time that CD31 has been reported to
bemobilized by either hyperbaric air or hyperbaric oxygen, it is not
unexpected, given that CD31 is associated with endothelial function
much like CD34 and CD133. It was beyond the scope of this research
project to determine if these mobilized CD31 stem cells participate in
wound healing and angiogenesis. Future research testing the eect of
hyperbaric air mobilized CD31 stem cells on healing, angiogenesis,
and atherosclerotic disease states would beprudent.
Our results also revealed an interesting relationship between SPCs
expressing CD45
dim
/CD34
+
/CD133
−
and those SPCs expressing CD45
dim
/
TABLE2 Summary of cell type frequency after initial and intermittent
exposure detected by flow cytometry.
CD45dim CD45+CD45−
CD45dim =,=,=,=
CD45+=,=,=,=
CD45_=,=,↓,=
CD31_ CD105+=,=,=,= =,=,=,= =,=,=,=
CD31+ CD105_=,=,↑,↑=,=,↓,↓=,=,=,=
CD31+ CD105+=,=,=,= =,=,=,= =,=,=,=
CD31_ CD105_=,=,=,= =,=,=,= =,=,=,=
CD34_ CD133+=,=,↓,↓=,=,↓,↓=,=,=,=
CD34+ CD133_=,=,↑,↑=,=,↑,↑=,=,=,=
CD34+ CD133+=,=,=,= =,=,=,= =,=,=,=
CD34_ CD133_=,=,=,= =,=,=,= =,=,=,=
Results from four time points represented by change symbol separated by commas.
e = symbol represents no change in frequency, the ↓ symbol represents a signicant
reduction in frequency, and the ↑ symbol indicates a signicant mobilization. e rst time
point is the control point taken prior to exposure. e second is taken directly aer the rst
exposure. e third timepoint was taken just prior to the 10th and nal exposure and the
fourth time point is the frequency 3 days aer cessation of exposures.

MacLaughlin et al. 10.3389/fneur.2023.1192793
Frontiers in Neurology 06 frontiersin.org
CD34
−
/CD133
+
. CD133+ SPCs are hematopoietic precursors to CD34
+
and almost all hematopoietic pluripotent and committed stem cells in
colony-forming assays express CD34+ (36). In this study wefound that
CD34+ SPCs increased while at the same time CD133+ SPCs decreased.
Wehypothesize that the exposure to intermittent hyperbaric air may
mobilize CD133
+
from bone marrow, and also play a role in the
dierentiation of the CD133+ primitive hematopoietic precursor into the
CD34
+
SPC. However, it has also been shown that CD133
+
have a high
capacity to dierentiate into other cell types including broblasts,
hepatocytes, and neural cell-types (35). Unfolding the explanation for the
signicant reduction in the expression of CD45
dim
/CD34
−
/CD133
+
expressing SPCs holds exciting potential.
Previous research has shown that breathing 100% oxygen at 2.4
atmospheres absolute mobilized stem/progenitor cells at a signicantly
greater rate than 2.0 Atmospheres Absolute suggesting a dose eect (41).
HBOT at 2.0 ATA (P
I
O
2
of 1426 mmHg and P
I
N
2
of 0 mmHg) resulted in
a two-fold mobilization of CD34
+
SPCs aer 1 exposure and an eight-fold
increase aer 20 exposures (28). In this study using 1.27 ATA of
hyperbaric air (P
I
O
2
of 189 mmHg and P
I
N
2
of 706 mmHg) we saw a
two-fold increase in SPC mobilization just prior to the 9th exposure. is
supports the hypothetical relationship between oxygen dose and stem
cell mobilization.
Another aim of this study was to determine if the stem progenitor
cell mobilization was durable. Because the stem progenitor cell
mobilization increased another fold following the end of exposures a
durable eect is likely.
Interestingly, wealso found changes in cells expressing CD45
+
cell
subtypes. ese changes were remarkably similar to changes in the
CD45dim population. CD45+ is expressed on all hematopoietic cells,
including HSCs and osteoclasts, which are of hematopoietic origin
(42), and is known as a pan-leukocyte marker.
Macrophage-derived chemokine (MDC) expressed in venous blood
signicantly decreased prior to the ninth exposure and returned to
pre-exposure levels 72 h following the nal exposure. e expression of
MDC is increased in idiopathic pulmonary brosis (43) and elevated
following resuscitation of patients aer hemorrhage. Macrophage-derived
chemokine may provide a therapeutic strategy to mitigate this
inammatory response (44). MDC is also thought to serve as a marker of
pharmacological therapy response in Major Depressive Disorder (45).
Changes in barometric and hydrostatic pressure may be a
mechanism of hyperbaric therapy. Small changes in atmospheric
pressure elicit responses in many organisms (46). However the
mechanism(s) is poorly understood. One possibility is that pressure is
sensed through hydrostatic compression of heterogeneous structures.
For instance, cells in suspension, including platelets (47, 48) and cartilage
cells (49, 50), respond to small changes in pressure ostensibly through
this mechanism. Another possibility is that cellular structures may
produce shear and strain through dierential compression. Microtubules,
actin and other cytoskeletal proteins respond to this type of local
mechanical stresses (51, 52). It should also benoted that certain animals
appear to respond to changes in atmospheric (air) pressure. For example,
pigeons exhibit changes in heart rate in response to changes of about
1 mbar, which is a typical daily uctuation in barometric pressure. But in
such cases, it is unclear how barometric pressure changes are sensed (53).
It is probable that changes in pressure have eects on human physiology.
Another possible mechanism ultimately resulting in stem cell
mobilization in this experiment is a progressive accumulation of
endogenous anti-oxidants at the cellular level. ese antioxidants accrue
in response to repeated exposure to a hyperoxic environment. e result
is an up-regulation of the Hypoxia-inducible factor 1α (HIF-1α)
transcription factor activity (54–60). is hypothetical mechanism is
best described as the Normoxic Hypoxic Paradox. is mechanism is
best characterized by an increase in reactive oxygen species (ROS) due
to the hyperoxic cellular environment. e increased cellular ROS
creates an imbalance of ROS/scavenger antioxidants ratio. e increased
ROS molecules initially hydroxylate most of the HIF-1α, facilitating
ubiquitination and degradation of most of the HIF-1α subunits.
However, it is postulated that an adaptive response to repeated
hyperoxia, increases the production of scavengers in proportion to the
increased ROS generation. e ROS/scavenger ratio gradually becomes
balanced in the hyperoxic environment. However, when the hyperoxic
exposure ends and the cell returns to a normoxic state, the ROS/
scavenger ratio is again imbalanced, but this time with more scavengers
than ROS, which produces a reduced ROS environment. Less ROS leads
to an increase in HIF-1α, initiating HIF transcription factor activity,
resembling a hypoxic state, but in a normoxic environment. In this
study CD34+/CD133- expression trended downward directly following
the initial hyperbaric air exposure, but increased signicantly prior to
the tenth exposure. e opposite was true with CD34
-
/CD133
+
. ese
results support the hypothetical Normoxic Hypoxic Paradox mechanism.
While our study clearly showed that the hyperbaric air dose that
was used as a placebo mobilizes proangiogenic and hematopoietic
stem progenitor cells and likely has a therapeutic physiologic eect
similar to hyperbaric oxygen therapy (29, 61–63), it was beyond the
scope of this study to determine clinical signicance. Hopefully our
results will generate renewed interest in hyperbaric air and future
studies will investigate both mechanism and clinical outcomes.
Limitations
e major limitation of our study is a relatively small sample size.
Future directions
It should not beoverlooked that although weincreased the
oxygen partial pressure and presume that oxygen is the active
element in hematopoietic and pro-angiogenic stem progenitor
mobilization, the partial pressure of nitrogen and remaining trace
gasses are also increased. Nitrogen is not an inert element and
can form five potential oxidation states and three potential
reduction states with various levels of reactivity (64). Recent
evidence suggests that intracellular reactive oxygen and nitrogen
species play an important role in intracellular signaling cascades
(65). However, little is known about the effect of nitrogen and
trace gasses on SPC mobilization or cytokine, chemokine and
growth factor modulation. Increased nitrogen and trace gasses as
a stimulator of SPC mobilization and chemokine modulation is a
possible mechanism in this research project and an exciting idea
to explore in future research.
It is very possible that stem cell mobilization by hyperbaric air
provides an additional modality to heal injuries. Because of its reduced
cost of delivery, it may prove benecial in developing nations and in
underserved populations. Hyperbaric air also increases safety because
the oxygen partial pressure is relatively low. e low oxygen partial

MacLaughlin et al. 10.3389/fneur.2023.1192793
Frontiers in Neurology 07 frontiersin.org
pressure in hyperbaric air may also provide increased therapeutic value
by not overloading the cerebral energy metabolism balance (66, 67).
Finally, our data suggests that the therapeutic dose of oxygen
begins at a much smaller partial pressure than previously thought and
adds a new data point on the initial portion of the hormetic curve of
oxygen dose.
Although wefound that intermittent small increases in hyperbaric
air pressure mobilized stem cells, this study should not betaken as an
endorsement of the use of intermittent hyperbaric air for any purpose
other than the indications approved by the FDA.
Why are these ndings important? First, prior to this research it was
not known that breathing a small increase in hyperbaric air would
mobilize stem progenitor cells. is knowledge could bean important
low-cost healthcare option when hyperbaric oxygen is not available,
especially in underserved populations, remote areas, and developing
nations. Second, because the technology is lightweight and portable, it is
hypothetically possible to beused when transporting combatants and
civilians out of a warzone to extend the viability of damaged tissue and to
reduce exacerbating gas embolism when air-liing by high altitude ights
is required. Finally, this research refutes the ndings of a placebo eect in
the decades-old use of slightly pressurized room air as a placebo in
hyperbaric oxygen research.
Conclusion and impact
In this study wedemonstrate for the rst time that intermittent
exposure to ostensibly insignicant pressures of hyperbaric air
mobilizes stem progenitor cells in a similar manner to that seen in
isobaric hyperoxia and hyperbaric oxygen therapy (21, 28, 29, 41).
Wealso establish that the stem progenitor cell mobilization is durable.
This research reveals that hyperbaric air, even at an ostensibly
insignificant dose, has significant effects on human physiology,
is not a placebo, and should beconsidered as an active physiologic
intervention. Its use as a pharmaceutical should
beinvestigated further.
Although this study did not test for clinical results, clinical
outcomes of hyperbaric air have been reported in similar
hyperbaric air studies (6–8). This study supports the data, but
refutes the conclusions in those studies by revealing a mechanism
of action for the clinical improvements reported in the hyperbaric
air group in those studies. Much more work is needed to develop
protocols of hyperbaric air dose that provide the maximum
therapeutic benefit.
ese ndings substantiate the need for testing hyperbaric air doses
prior to using hyperbaric air as a placebo in scientic investigations.
ese ndings also substantiate the urgent need for reevaluation of
ndings in historical studies using hyperbaric air placebos. Our ndings
suggest that these historical placebo-controlled studies were not placebo-
controlled studies. Paradoxically, our ndings indicate that they were
dose studies and the ndings of a “placebo eect” or “participation eect”
are inherently awed. e “ndings” and “conclusions” in studies using
hyperbaric air as a placebo should bereevaluated from a dose study
perspective. Finally, wehope the ndings in this study will persuade the
medical societies around the world to consider reevaluation of their
denition of hyperbaric medicine to include nominal hyperbaric air.
Looking back at the “Hyperbaric Air” work of Henshaw, Simpson
and Cunningham, our ndings support their reports.
Data availability statement
e original contributions presented in the study are included in
the article/supplementary material, further inquiries can bedirected
to the corresponding author.
Ethics statement
e studies involving human participants were reviewed and
approved by the University of Wisconsin–Madison. e patients/
participants provided their written informed consent to participate in
this study.
Author contributions
KM and GB: ideas conception. KM, GB, RB, and ME: study and
experiments design. KM, JM, RB, JL, and MM: experiments perform,
data acquisition, and analysis. GB and RB: guidance and critical
feedback on data acquisition and analysis. RB and ME: project
supervision. KM: manuscript writing. All authors provided critical
feedback and contributed to the nal version of manuscript.
Funding
is work was supported by the Foundation for the Study of
Inammatory Disease and the International Hyperbaric Association.
Acknowledgments
e authors would like to thank the study participants for their time
and dedication to complete this study. anks also goes to Caleb and
Sidney Gates, Dr. Rob Beckman, Dr. Vivek Balasubramanium, Dr. Barb
Bendlin, Dr. Robert Stone, Dr. William Schrage, Dr. Paul Harch, Dr.
Phillip James, Dr. Don Wollheim, Dr. Donata Oertel, Dr. Awni Al-Subu,
Dr Kara Goss, Dr. Arij Beshish, Dr. Andrew Watson, Dr. Xavier Figueroa,
Dr. Daphne Denham, Tom Fox, Freya Christensen, the University of
Wisconsin CCC Flow Cytometry Lab, Dagna Sherrar, Lauren
Nettenstrom, Kathryn Fox and Zach Sterneson, the Univeristy of
Wisconsin-Madison Department of Pediatrics and Gradaute School, Dr.
Ellen Wald, Dr. James Gern, and Kim Stevenson for the support.
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.

MacLaughlin et al. 10.3389/fneur.2023.1192793
Frontiers in Neurology 08 frontiersin.org
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