Response of Circulating Inflammatory Markers to Intermittent Hypoxia-Hyperoxia Training in Healthy Elderly People and Patients with Mild Cognitive Impairment

Abstract

Intermittent hypoxia-hyperoxia training (IHHT) is a non-pharmacological therapeutic modality for management of some chronic- and age-related pathologies, such as Alzheimer’s disease (AD). Our previous studies demonstrated significant improvement of cognitive function after IHHT in the patients with mild cognitive impairment (MCI). The present study further investigated the effects of IHHT on pro-inflammatory factors in healthy elderly individuals and patients with early signs of AD. Twenty-nine subjects (13 healthy subjects without signs of cognitive impairment syndrome and 16 patients diagnosed with MCI; age 52 to 76 years) were divided into four groups: Healthy+Sham (n = 7), Healthy+IHHT (n = 6), MCI+Sham (n = 6), and MCI+IHHT (n = 10). IHHT was carried out 5 days per week for 3 weeks (total 15 sessions), and each daily session included 4 cycles of 5-min hypoxia (12% FIO2) and 3-min hyperoxia (33% FIO2). Decline in cognitive function indices was observed initially in both MCI+Sham and MCI+IHHT groups. The sham training did not alter any of the parameters, whereas IHHT resulted in improvement in latency of cognitive evoked potentials, along with elevation in APP110, GDF15 expression, and MMP9 activity in both healthy subjects and those with MCI. Increased MMP2 activity, HMGB1, and P-selectin expression and decreased NETs formation and Aβ expression were also observed in the MCI+IHHT group. There was a negative correlation between MoCA score and the plasma GDF15 expression (R = −0.5799, p < 0.05) before the initiation of IHHT. The enhanced expression of GDF15 was also associated with longer latency of the event-related potentials P330 and N200 (R = 0.6263, p < 0.05 and R = 0.5715, p < 0.05, respectively). In conclusion, IHHT upregulated circulating levels of some inflammatory markers, which may represent potential triggers for cellular adaptive reprogramming, leading to therapeutic effects against cognitive dysfunction and neuropathological changes during progression of AD. Further investigation is needed to clarify if there is a causative relationship between the improved cognitive function and the elevated inflammatory markers following IHHT.

Keywords: Alzheimer’s disease; cognitive Impairment; hypoxia inducible factor 1; inflammation; intermittent hypoxia; neutrophil extracellular traps.

Figure 1

Illustrative description of the protocol for intermittent hypoxic-hyperoxic training (IHHT) sessions. Abbreviation: FiO₂, Fraction of inspired oxygen levels.

Figure 2

Effects of IHHT on cognitive parameters in healthy elderly controls and patients with mild cognitive impairment (MCI). The measurements were conducted at three time-points: Initial—the day before the initiation of sham or IHHT course; 1 day—one day after the termination of 3-week IHHT sessions; 1 month—one month after the termination of 3-week IHHT sessions. Bar 1—Healthy+Sham; Bar 2—Healthy+IHHT; Bar 3—MCI+Sham; and Bar 4—MCI+IHHT. (A) shows the Montreal Cognitive Assessment test (MoCA); (B,C) indicate the latency of N200 and P300 peaks of cognitive evoked potential. Data are presented as mean ± standard deviation (SD). Abbreviations: R_F—significant difference (p < 0.05) compared to Initial by the Friedman test, R_W—significant difference (p < 0.05) compared to Initial by the Wilcoxon test, R_MW—significant difference (p < 0.05) between groups by the Mann–Whitney U test.

Figure 3

Effects of IHHT on Alzheimer disease-related markers in healthy elderly controls and patients with mild cognitive impairment (MCI). The measurements were conducted at three time-points: Initial—the day before the initiation of sham or IHHT course; 1 day—one day after the termination of 3-week IHHT sessions; 1 month—one month after the termination of 3-week IHHT sessions. Bar 1—Healthy+Sham; Bar 2—Healthy+IHHT; Bar 3—MCI+Sham; and Bar 4—MCI+IHHT. (A) shows expression levels of amyloid beta 1-42 (Aβ) in platelets; (B,C) indicate expression of APP130 and APP110 in platelets; and (D) shows the ratio of APP110/APP130 (amyloid precursor protein isoforms 110/130) in platelets. Data are presented as mean ± standard deviation (SD). Abbreviations: R_F—significant difference (p < 0.05) compared to Initial by the Friedman test, R_W—significant difference (p < 0.05) compared to Initial by the Wilcoxon test, R_MW—significant difference (p < 0.05) between groups by the Mann–Whitney U test.

Figure 4

Effects of IHHT on circulating inflammatory markers in blood samples collected from healthy elderly controls and patients with mild cognitive impairment (MCI). The measurements were conducted at three time-points: Initial—the day before the initiation of sham or IHHT course; 1 day—one day after the termination of 3-week IHHT sessions; 1 month—one month after the termination of 3-week IHHT sessions. Bar 1—Healthy+Sham; Bar 2—Healthy+IHHT; Bar 3—MCI+Sham; and Bar 4—MCI+IHHT. (A) shows expression levels of High Mobility Group Box Protein 1 (HMGBP1b); (B) shows p-selectin (Psel); (C) indicates cytochrome C (cytC); (D) shows Growth Differentiating Factor 15 (GDF15); (E,F) indicate activity of matrix metalloproteinases 2 and 9 (MMP2 and MMP9); (G) shows neutrophil extracellular traps non stimulated (NETns); (H) shows neutrophil extracellular traps stimulated by phorbol miristate acetate (NETst); (I) shows expression of tumor necrosis factor α (TNFα); and (J) shows expression of hypoxia inducible factor 1α antisense long noncoding RNA (HIF1α-AS1). Data are presented as mean ± standard Deviation (SD). Please note that the data are missing due to a technical failure in RNA isolation for the assessment of HIF1α-AS1 at 1 month after the IHHT timepoint in the MCI+Sham group. Abbreviations: R_F—significant difference compared to Initial by the Friedman test, R_W—significant difference (p < 0.05) compared to Initial by the Wilcoxon test, R_MW—significant difference (p < 0.05) between groups by the Mann-Whitney U test.

Figure 4

Effects of IHHT on circulating inflammatory markers in blood samples collected from healthy elderly controls and patients with mild cognitive impairment (MCI). The measurements were conducted at three time-points: Initial—the day before the initiation of sham or IHHT course; 1 day—one day after the termination of 3-week IHHT sessions; 1 month—one month after the termination of 3-week IHHT sessions. Bar 1—Healthy+Sham; Bar 2—Healthy+IHHT; Bar 3—MCI+Sham; and Bar 4—MCI+IHHT. (A) shows expression levels of High Mobility Group Box Protein 1 (HMGBP1b); (B) shows p-selectin (Psel); (C) indicates cytochrome C (cytC); (D) shows Growth Differentiating Factor 15 (GDF15); (E,F) indicate activity of matrix metalloproteinases 2 and 9 (MMP2 and MMP9); (G) shows neutrophil extracellular traps non stimulated (NETns); (H) shows neutrophil extracellular traps stimulated by phorbol miristate acetate (NETst); (I) shows expression of tumor necrosis factor α (TNFα); and (J) shows expression of hypoxia inducible factor 1α antisense long noncoding RNA (HIF1α-AS1). Data are presented as mean ± standard Deviation (SD). Please note that the data are missing due to a technical failure in RNA isolation for the assessment of HIF1α-AS1 at 1 month after the IHHT timepoint in the MCI+Sham group. Abbreviations: R_F—significant difference compared to Initial by the Friedman test, R_W—significant difference (p < 0.05) compared to Initial by the Wilcoxon test, R_MW—significant difference (p < 0.05) between groups by the Mann-Whitney U test.

Figure 4

Effects of IHHT on circulating inflammatory markers in blood samples collected from healthy elderly controls and patients with mild cognitive impairment (MCI). The measurements were conducted at three time-points: Initial—the day before the initiation of sham or IHHT course; 1 day—one day after the termination of 3-week IHHT sessions; 1 month—one month after the termination of 3-week IHHT sessions. Bar 1—Healthy+Sham; Bar 2—Healthy+IHHT; Bar 3—MCI+Sham; and Bar 4—MCI+IHHT. (A) shows expression levels of High Mobility Group Box Protein 1 (HMGBP1b); (B) shows p-selectin (Psel); (C) indicates cytochrome C (cytC); (D) shows Growth Differentiating Factor 15 (GDF15); (E,F) indicate activity of matrix metalloproteinases 2 and 9 (MMP2 and MMP9); (G) shows neutrophil extracellular traps non stimulated (NETns); (H) shows neutrophil extracellular traps stimulated by phorbol miristate acetate (NETst); (I) shows expression of tumor necrosis factor α (TNFα); and (J) shows expression of hypoxia inducible factor 1α antisense long noncoding RNA (HIF1α-AS1). Data are presented as mean ± standard Deviation (SD). Please note that the data are missing due to a technical failure in RNA isolation for the assessment of HIF1α-AS1 at 1 month after the IHHT timepoint in the MCI+Sham group. Abbreviations: R_F—significant difference compared to Initial by the Friedman test, R_W—significant difference (p < 0.05) compared to Initial by the Wilcoxon test, R_MW—significant difference (p < 0.05) between groups by the Mann-Whitney U test.

Figure 5

Summarized effects of IHHT on neurological and circulating parameters in healthy elderly participants (in green oval) and patients with MCI (in pink oval). Abbreviations: IHHT, intermittent hypoxia-hyperoxia training; MCI, mild cognitive impairment; Hyp, marker of hypoxia; Cog, cognitive related parameters; AD, Alzheimer disease related parameters; Inf, inflammation-related parameters; MoCA, Montreal Cognitive Assessment test; N200 and P300, latencies of N200 and P300 peaks of cognitive evoked potential; Aβ, amyloid beta 1-42 in platelets; APP110, amyloid precursor protein isoform 110 in platelets; HMGBP1b, High Mobility Group Box Protein 1b; P-sel, p-selectin; CytC, cytochrome C; GDF15, Growth Differentiating Factor 15; MMP2 and MMP9, matrix metalloproteinases 2 and 9; NETs, neutrophil extracellular traps; TNFα, tumor necrosis factor α; HIF1α-AS1, hypoxia inducible factor 1α antisense long noncoding RNA.