The influence of acute, chronic and lifelong hypoxia on the functional and structural integrity of the neurovascular unit

Abstract

Background. The current thesis adopts a whole-body, integrated approach to investigate the impact of acute, chronic and life-long exposure to hypoxia on the functional and structural integrity of the neurovascular unit (NVU).

Methods. Experimental Study 1: In a randomised, single-blind cross-over design, 12 healthy, physically active male participants (age, 23 ± 2 yrs; BMI, 25 ± 4 kg/m²) were exposed to 6 hours of normoxia (F_IO₂ = 0.21) and 6 hours of acute normobaric hypoxia (F_IO₂= 0.12) on separate visits. Experimental measures were conducted at baseline (0 h) and following 6-hours of each exposure. Experimental Study 2: In a repeated-measures, cross-sectional study, nine native lowlanders (28 ± 8 yrs, 24.3 ± 1.7 kg∙m-²) were examined at sea-level (normoxia) and following 14 days of high-altitude (HA) acclimatisation to 4,300 m in Cerro De Pasco, Peru (chronic HA hypoxia). Age- and BMI-matched healthy native highlanders of the Altiplano (~4300 m) (n = 9, 27 ± 7 yrs, 26.1 ± 6.0 kg/m²) were also examined (life-long HA hypoxia). Experimental measures for Study 1 and 2: Venous blood samples were assayed for biomarkers specific to oxidative-nitrosative stress (OXNOS; ascorbate free radical [A^•−] and nitric oxide [NO] metabolites via electron paramagnetic resonance spectroscopy and ozone-based chemiluminescence, respectively) and structural integrity of the NVU (Study 1: S100ß and neuron-specific enolase [NSE]; Study 2 only: S100ß, NSE, ubiquitin carboxy-terminal hydrolase L1 [UCH-L1], glial fibrillary acidic protein [GFAP], neurofilament light-chain [NF-L] and total-tau [T-Tau] determined via automated clinical grade ELISA and Single Molecule Array technology). Cardiopulmonary variables included mean arterial pressure (MAP), heart rate (HR), stroke volume (SV), cardiac output (Q), peripheral oxygen saturation (SpO₂), minute ventilation (V̇E), partial pressure of end-tidal oxygen (P_ETO₂) and carbon dioxide (P_ETCO₂), haematocrit (Hct), haemoglobin (Hb), arterial oxygen content (CaO₂) and blood viscosity (Study 2 only). Duplex and transcranial Doppler ultrasonography were employed to measure extracranial (internal carotid artery blood flow [ICA_Q] and vertebral artery blood flow [VA_Q]) and intracranial (middle cerebral artery blood velocity [MCA_V] and posterior cerebral artery [PCA_V]) haemodynamics. Global cerebral blood flow (gCBF) was calculated and used to quantity global cerebral substrate delivery of oxygen (gCDO₂) and glucose (gCD_Glu). NVU function was examined via: 1, dynamic cerebral autoregulation (dCA) assessed using transfer function analysis during spontaneous oscillations in MAP and MCA_V/ PCA_V; and 2, neurovascular coupling (NVC) via PCA_V responses to repeated dark (eyes closed) and light (eyes open + visual stimulation) activation.

Results. Experimental Study 1: Hypoxia failed to alter A^•− , total plasma NO and total RBC NO. Owing to the reduction in SpO₂ and unaltered Hb, caO₂ was decreased in hypoxia. An increase in V̇E following 6 h of hypoxia facilitated an increase in P_ETO₂ and a reduction in P_ETCO₂ . In hypoxia, HR and Q̇ were elevated and MAP was reduced. ICA_Q and VA_Q were both elevated in hypoxia. Despite an increase in gCBF in hypoxia, gCDO₂ was lower in hypoxia and was unaffected by time. While ICA_Q and ICA DO₂ was reduced in hypoxia, VA_Q and VA DO₂ remained well preserved. gCD_Glu was elevated in hypoxia owing to increase ICA D_Glu and VA D_Glu. Hypoxia reduced dCA (lower VLF Phase in the MCA and PCA, increased VLF Gain in the PCA) but had no effect on NVC. S100β remained unchanged and NSE decreased in hypoxia. Experimental Study 2: Chronic HA hypoxia in lowlanders increased Hct, Hb and blood viscosity, restoring CaO₂ to baseline normoxic values. While highlanders were more polycythaemic, they were equally hypoxaemic albeit slightly less alkalotic, hypocapneac and hypertensive. Chronic HA hypoxia in lowlanders decreased A^•− and was accompanied by a reciprocal elevation in plasma and RBC NO bioavailability. Highlanders presented with similar levels of A^•− to lowlanders at sea-level, however NO bioavailability (nitrite [NO₂⁻] and S-nitrosothiols) was further elevated. Following chronic HA hypoxia in lowlanders, gCBF, gCD_Glu, gCD O₂ and NVC were comparable to normoxic values and equivalent to those observed in highlanders. Although MCA/PCA phase estimates were also comparable to sea-level following chronic HA hypoxia in lowlanders, gain metrics were consistently lower in the highlanders. Chronic HA hypoxia increased NF-L and decreased T-tau whereas UCHL-1, NSE, GFAP and S100ß did not change. With the exception of NSE and GFAP, all NVU biomarkers were lower in highlanders (lifelong HA hypoxia) compared to lowlanders following chronic HA hypoxia.

Discussion: By utilising an integrated approach, this thesis examined to what extent acute, chronic and life-long hypoxia alters the functional (regional CBF substrate delivery, dCA, and NVC) and structural (BBB permeability and neuronal-axonal damage) integrity of the NVU, whilst examining potential mechanisms (OXNOS) underlying these changes. Contrary the original working hypothesis, acute hypoxia was associated with marked reductions in global substrate delivery and diminished anterior and posterior cerebral autoregulatory capacity, but it was not associated with changes in OXNOS and further, did not translate to impaired NVC, increased BBB permeability or provide any evidence for on-going neuronal-axonal damage. Study 2 supported the working hypothesis whereby following chronic HA hypoxia, it would appear that global substrate delivery and NVC remained well-preserved and dCA improved. Following life-long HA hypoxia in Andean highlanders, the consistently lower levels of S100ß, NF-L and T-Tau observed indicate that the NVU is structurally ‘tighter’ when compared with lowlanders who present with evidence of on-going axonal damage. That global cerebral perfusion and substrate delivery remained well preserved and the lower (LF) gain observed in the anterior/posterior circulation highlights improved pressure-flow coupling (potentially mediated by elevated systemic concentrations of basal plasma and RBC NO), this may indicate that the highlander brain is better equipped to buffer perfusion in response to rapid increases in BP, further ‘bolstering’ the NVU against the potentially damaging effects of BBB disruption. Evidence of such neuroprotection may be presented in the form of the comparable NVC responses between lowlanders at sea-level and highlanders. Further study of the mechanisms governing NVU adaptation in the hypoxia-tolerant human may help inform the pathophysiology and treatment of neurodegenerative diseases notwithstanding hypoxia-related illnesses such as stroke, heart failure, lung disease and cancer.

Abbreviations
A^•−– Ascorbate free radical
ACA – Anterior cerebral artery
AMS – Acute mountain sickness
ATP – Adenosine tri-phosphate
β93 Cys – β93 cysteine residue
BBB – Blood-brain-barrier
BMI – Body mass index
BOLD – blood oxygenation level-dependant
CA – Cerebral autoregulation
cAMP – Cyclic adenosine monophosphate
CaO₂– Arterial oxygen content
CBF – Cerebral blood flow
CDO₂ – Cerebral oxygen delivery
cGMP – Cyclic guanosine monophosphate
CMRO₂ – Cerebral metabolic rate of oxygen
CSF – Cerebrospinal fluid
CVR – Cerebrovascular reactivity
DO₂ – Delivery of oxygen
EET – epoxyeicosatrienoic acid
eNOS – Endothelial nitric oxide synthase
F_ICO₂ – Fraction of inspired carbon dioxide
F_IO₂– Fraction of inspired oxygen
GABA - γ-Aminobutyric acid
gCBF – Global cerebral blood flow
gCD_Glu – Global cerebral delivery of glucose
gCDO₂ – Global cerebral delivery of oxygen
HA – High-altitude
Hb – Haemoglobin
HbFe(II) – Ferrous haemoglobin
HbFe(III) – Ferric haemoglobin or methaemoglobin
HCO₃^- – Concentration of bicarbonate
Hct – Haematocrit
I₃^- – Triiodide
ICA – Internal carotid artery
ICAv – Internal carotid artery blood velocity
ICP – Intracranial blood pressure
iNOS – Inducible nitric oxide synthase
L-NMMA – N^G monomethyl-L-arginine
MCA – Middle cerebral artery
MCAv – Middle cerebral artery blood velocity
MHz – Megahertz
MLCK – Myosin light chain kinase
mmHg – Milimettres of mercury
MRI – Magnetic resonance imaging.
NaOH – Sodium hydroxide
NF-L – Neurofilament Light Chain
NINDS – National Institute of Neurological Disorders and Stroke
NIH – National Institutes of Health
nNOS – Neuronal nitric oxide synthase
NO – Nitric oxide
NO₃⁻ – Nitrate
NO₂⁻ – Nitrite
NVC – Neurovascular coupling
NVU – Neurovascular unit
N₂O – Nitrous oxide
OH^- – Hydroxide
PaCO₂ – Partial pressure of arterial carbon dioxide
PaO₂ – Partial pressure of arterial oxygen
PCA – Posterior cerebral artery
PCAv – Posterior cerebral artery blood velocity
PET – Positron emission tomography
P_ETO₂ – Partial pressure of end-tidal oxygen
P_ETCO₂ – Partial pressure of end-tidal carbon dioxide
PKA – Protein kinase A
PO₂ – Partial pressure of oxygen
PjvO₂ – Partial pressure of jugular venous oxygen
PvCO₂ – Partial pressure of venous carbon dioxide
PvO₂ – Partial pressure of venous oxygen
ROS – Reactive oxygen species
RSNO – S-nitrosothiols
SL – Sea-level
SMCs - Smooth muscle cells
SNA – Sympathetic nervous activity
SNO-Hb – S-nitrosohemoglobin
SpO₂ – Peripheral oxyhaemoglobin saturation
TCD – Transcranial Doppler ultrasound
TPR – Total peripheral resistance
T-Tau – Total tau
VA – Vertebral artery
VAv – Vertebral artery blood velocity
V̇_E – Minute ventilation 

Date of Award	2024
Original language	English
Supervisor	Damian Bailey (Supervisor), Christopher Marley (Supervisor) & Lewis Fall (Supervisor)