Our goal in this study was to identify the systemic and temporal changes in gene expression occurring in blood during the exposure and recovery from hypoxia to identify novel genes to be used as possible reference genes (biomarkers) detectable by qPCR in small samples of peripheral blood. Since all blood cells were used rather than sorted by cell types, we cannot attribute contribution of gene changes to specific peripheral blood cell type or types, and thus discuss the changes in gene expression and analyses as an effect of CH present in the whole peripheral blood.
Out of 230 differentially regulated transcripts found to be differentially regulated, we did not identify any differentially expressed transcripts from the list of 86 HIF-regulated genes that have been detected in a stringent meta-analysis across 6 cell types 
. However, we did identify 5 genes differentially expressed in liver from mice exposed to CH 
(solute carrier family 16, B-cell translocation gene 3, testis-specific protein; Y-encoded-like 1, CD36 antigen and preimplantation protein 4). Another comparison of our differentially regulated genes with a set of genes derived from bone marrow of mice exposed to intermittent hypoxia also revealed only 15 genes to be in common as shown in Supplemental Table S3
. These comparisons show that HIF may not directly regulate the expression of our 230 transcripts during CH and support the evidence that while HIF is important during the acute phase response during hypoxia to regulate local and systemic mechanisms that underpin oxygen sensing 
, it may not play a major role in the chronic phase of acclimatization to CH. However, comparisons with 11 other studies who have studied the effects of hypoxia across different species and tissues using differing experimental protocols for hypoxia exposure revealed no genes in common with our expression profile suggesting its specificity to tissue type and condition studied 
As a systemic response for acclimatization to CH, we observed a significant loss of BW over the period of CH exposure, reaching its lowest level at the end of the CH protocol (CH-14), confirming previous reports 
. However, during recovery, the BW rapidly recovered to similar levels as before CH exposure. Hypoxia-induced BW loss has been extensively reported 
. The mechanisms involving BW loss are thought to be mediated mainly by carbohydrate and lipid metabolisms. The glucose metabolism has been extensively studied and is considered a major energy pathway involved in the response to hypoxia 
. Subjects lost weight and did not increase their blood sugar level despite drinking 100 grams of glucose after a 17 days-expedition to the Chilean Andes exposed to high altitude (5340 MASL) 
. CH induces the expression of glucose transporter (GLUT) types 1 and 4 in rat cardiac and skeletal muscles 
and GLUT1 in the brain blood barrier 
, suggesting that the lower plasma glucose induced by CH leads to higher expression and activity of GLUT in several tissues. Finally, hypoxia inhibits liver gluconeogenesis, subsequent to a reduction in phosphoenolpyruvate carboxykinase activity 
. Together, these results suggest that the loss of BW during CH exposure may be attributed to increased carbohydrate catabolism.
A novel mechanism for BW regulation during hypoxia may involve the leptin pathway, which is a regulator of BW and lipid metabolism during hypoxia. Leptin is an obese (ob) gene product and its mature form contains a 167 amino-acid peptide hormone 
. It is produced in adipose tissue 
, placenta 
, stomach 
, muscle tissue 
, and cerebral adenohypophysis and hypothalamus 
. The action of leptin is mediated by 6 different isoforms of the receptor present in the hypothalamus 
, regulating BW by suppressing food intake and increasing the energy expenditure, glucose and fatty acid metabolism, hematopoiesis and immune system 
. The ob gene contains a HRE site that can be bound by HIF in its 5′-untranslated region inducing up-regulation of leptin expression under hypoxic challenges 
. Recently, Baze et al.
showed that chronic exposure to hypobaric hypoxia (4500 MASL) increases the leptin receptor expression in liver samples 
. In concordance with those results, rats exposed during 90 days at 5500 MASL showed an increase of lipid metabolism in both liver and skeletal muscle 
. These results suggest that fatty acid catabolism, hematopoiesis and energy expenditure are important factors for acclimatization to hypoxia and that the leptin pathway may play a profound role as a part of the mechanism for BW regulation noted during CH acclimatization.
We observed that CH and REC treatments had significant effects on classical peripheral blood parameters. The hematocrit level increased from 40% at NX to 54% and 74% at CH-7 and CH-14, respectively. The levels of hematocrit from CH-14 only returned to similar levels of NX-0 normal levels after 14 days of REC. Interestingly, the hematocrit at REC-7 presented similar levels as CH-7, suggesting that the turnover of erythrocytes is similar during the waxing and waning of hematopoeisis during CH and REC. It is well established that hypoxia induces EPO expression under either physiological or pathophysiological conditions, resulting in an increase of reticulocyte, hematocrit and hemoglobin concentration 
. While the relative numbers of reticulocytes were noted to be increased at CH-3 compared to NX-0, the increase became significant at CH-7, but did not continue to increase after 14 days of CH compared to CH-7. Rather, the expression level decreased, but still remained above normoxic level. The percentage of reticulocytes showed similar levels as at NX-0 once returned to REC. Our results are in concordance with studies performed in humans exposed to either intermittent or continuous CH (7 days, FiO2
15.4%) which reported an increase of the relative amount of reticulocytes on the 5th
. The hypoxia-induced increase in reticulocytes is also known to occur during prolonged hypoxic exposure. After a 21-day expedition to the Andes with continuous exposure above 4000 MASL (maximum of 6768 MASL), subjects submitted to four hours in a hypobaric chamber (4500 MASL, 443 mmHg) showed a new increase in the number of reticulocytes, as well as in the percentage of red blood cells 
. This result shows the capacity of colony forming unit-erythroid to response to new hypoxic challenge and continue to form new reticulocytes. 
. This hypoxia-mediated reticulocytosis phenomenon has also been reported in animals, with a slightly different time-course response. A study using rats exposed to a hypobaric hypoxia protocol (21 days at 0.5 atm; PB 380 mm Hg) showed an increase in the relative number of reticulocytes on day 3, reaching the maximum on day 7, before returning to normal levels on day 14. However, the reticulocytes reached a new peak on day 21 of the protocol 
. These results suggest a negative feedback on the hypoxia-induced reticulocytes formation, which reaches its maximum after 5–7 days of hypoxia before decreasing to normal levels, as we have described here.
One of the major aims of our study was to analyze the expression profile to identify and validate novel genes that could be used as reference genes (biomarkers) of CH and REC. Towards this goal we studied the expression levels of Spna1, Ubfd1 and Pycr1 as well as the previously characterized CD36 gene expression in detail. The pattern of differential expression of Spna1 paralleled changes in hematocrit during CH and REC, while Ubdf1 and Pycr1 genes were down- and up-regulated respectively, during the CH and REC periods. CD36 is a 88 KDa cell surface protein that belongs to an evolutionary conserved Class B scavenger receptors family 
. The expression of CD36 in erythroid cells is limited to reticulocytes, absent in mature erythrocytes 
and has been used as a marker for early erythroid cells 
. Acute hypoxia up-regulates CD36 in intrapulmonary arteries via activation of HRE mediated by HIF 
. In our microarray analysis, CD36 was up-regulated in mice peripheral blood during CH (fold-change 16.65) and then down-regulated during REC (fold-change -2.85). This pattern of expression was confirmed by qPCR in our extended time-point protocol 2, showing a significant increase in CD36 expression already after day 2 of CH and a marked down-regulation upon return to normoxia. The significant increase partially overlapped with the increased relative amount of reticulocytes on CH-7. This difference may be explained by the proliferation of reticulocytes induced by hypoxia 
. Nevertheless, the expression of CD36 was completely suppressed by REC, suggesting that reticulocyte proliferation activity was also reduced during REC.
Spna1 is a 280 KDa subunit of the spectrin cytoskeletal protein 
. Spectrin is a major constituent of red blood cell membranes and responsible for resistance to mechanical stress, cellular form and anchors for other proteins such as hemoglobin receptors and channels 
. Our results suggest that in whole blood samples, Spna1 expression is increased in response to CH. However its expression level declined during REC reaching NX levels, paralleling changes in hematocrit.
Ubfd1 is a novel polyubiquitin binding protein 
. Although hypoxia-induced changes in expression have not previously been described, analysis of primary data (GEO number GDS1807) in a study using a human lymphocyte cell line exposed to FiO2
0.1% for 29 hours demonstrated down-regulation of two transcripts (224878_at and 20568_at) of this gene 
. These data are in agreement with our demonstration that Ubfd1 expression is sensitive to CH and remained significantly down-regulated from the beginning of the hypoxic exposure until several days after the REC.
Pycr1 is a mitochondrial enzyme that converts pyrroline-5-carboxilic acid into proline in a NAD(P)H-dependent reaction 
. Pycr1 generates an oxidizing potential to stimulate the metabolism of glucose through the hexose monophosphate-pentose pathway and the Embden-Meyerhof pathway, an important pathway in erythrocytes 
. The increase of Pycr1 expression during CH suggests an increase of erythrocyte metabolism and glucose consumption in order to increase the amount of NAD(P)+
, not limiting the production of ATP via the anaerobic pathway of glycolysis. In astrocytes, 24 hours of hypoxia up-regulates Pycr1 expression and has been shown to be mediated by p53 
. In our profile, Pycr1 expression increased upon CH challenge, increased its expression further during CH and remained above the normoxic expression level even after 14 days of REC.
In conclusion, we describe the molecular signature for whole peripheral blood samples from mice challenged to CH and REC. The expression profile of genes separable into four distinct temporal categories (I-IV) using our criteria offers novel biomarkers to monitor a variety of physiological and pathological conditions associated with exposure to hypoxia and during recovery. While methods such as pulse oximetry or blood gas analysis are accurate and useful for detecting hypoxia as it occurs, they present limited utility in terms of being able to report prior occurrence of hypoxia or indeed recovery from prior hypoxic exposure. In contrast to using increased hematocrit levels as a marker for prior hypoxia exposure, the suggested gene-based biomarkers may be able to distinguish between chronic hypoxia and recovery from hypoxia with greater accuracy and independent of clinical states that often accompany hypoxia (e.g. volume dysregulation) which may falsely alter hematocrit levels. We believe that the novel biomarkers described here may be used to detect prior hypoxic exposure of up to 2 weeks duration either in isolation or as a combination (e.g. from Categories II & IV) analogous to the manner in which HbA1c is used to report the average blood glucose level over the previous month rather than just at the time of the test while managing patients with Diabetes. We believe that these potential novel biomarkers will be of great benefit to the research community and clinicians as it enables the detection of hypoxia in a post hypoxic state. However, we think it will be important to test the biomarkers identified here in a variety of animal models and clinical settings prior to their utilization.