Baseline Cardiovascular and Oximetric Parameters Across the Lifespan of Male Sprague Dawley Rats

doi:10.21203/rs.3.rs-5448976/v1

Human populations are experiencing unprecedented growth and longevity creating an urgent need for research into the pathologies, mechanisms, and dysfunctions of senescence. Invasive measurements and long-term control conditions for longitudinal studies are infeasible in humans, necessitating surrogate animal models. Rats have short lifespans (2–3 years) with translatable cardiovascular systems, and Sprague Dawley microcirculatory preparations are key to studying the oxygen transport mechanisms critical to aging. Here we present baseline physiological data of 61 male, Sprague Dawley rats at 3, 6, 12, 18, and 24 months of age. Anesthetized animals were surgically prepared for femoral arterial and venous cannulations, tracheal intubation, and exteriorization of the spinotrapezius muscle. Measurements included cardiovascular function, blood gases, and peripheral tissue interstitial oxygen tension (P_ISFO₂) using phosphorescence quenching microscopy. Intrinsic heart rates decreased with age without significant changes to blood pressure. Arterial oxygen tension declined 17% by 18 and 24 Months (p < 0.05) while p_ACO₂ and P_ISFO₂ were unchanged. Lactate was elevated at 12 and 18 Months along with an alkaline shift in blood pH. Heart rate and the decline in p_AO₂ without a rise in p_ACO₂ are conserved phenomena in human aging. The continuity of resting P_ISFO₂ despite an anaerobic shift in metabolism may be due to declining mitochondrial function and dysregulation of the vascular response to hypoxemia, which are also present in aged humans. These findings contribute to our understanding of age-related physiological changes and underscore the suitability of the Sprague Dawley rat model for aging research.

Aging

Sprag Dawley rat

Spinotrapezius muscle

Microcirculation

Oxygen

Phosphorescence Quenching Method

As humans and rodents age, their cardiovascular efficiency decreases significantly. From pulmonary gas exchange to cardiac output to blood flow distribution, progressive reductions in function erode effective oxygen delivery and metabolite clearance of respiring tissues. The functional consequences are well-described throughout human gerontology (for reviews on declining skeletal muscle function and mitochondrial metabolism in aging, see references (Brooks and Faulkner 1994, Hood, Memme et al. 2019, Amorim, Coppotelli et al. 2022), but deeper, mechanistic studies often require testable surrogates of healthy and pathological aging. Murine species with short life spans (~ 3 years), controllable living conditions, and translatable cardiopulmonary physiology (Papadimitriou, Xanthos et al. 2008) offer a platform for studying elements of aging.

There are three critical links in the oxygen supply chain: lungs, heart, and vasculature. Dysfunction of each comprises three of the top five causes of death in aged (> 65 years) humans—heart, chronic lower respiratory, and cerebrovascular diseases (Rana, Khan et al. 2021). Senescent pulmonary function manifests as increased alveolar dead space. Decreased thoracic elasticity reduces arterial oxygenation (p_AO₂), while arterial pCO₂ remains stable (Cerveri, Zoia et al. 1995, Sharma and Goodwin 2006). Insensitivity of p_ACO₂ to reduced p_AO₂ contributes to blunted ventilatory responses to hypoxemia and hypercapnia in the elderly (Peterson, Pack et al. 1981, Brischetto, Millman et al. 1984, Chapman and Cherniack 1987), which can exacerbate the development of pulmonary disease. Cardiac changes include ventricular remodeling (increased mass-to-volume ratio) and desensitization to sympathetic stimuli resulting in reduced cardiac output (Jakovljevic 2018) and lower maximum heart rates (Peters, Sharpe et al. 2020). There is evidence of reduced intrinsic heart rate as well (Christou and Seals 2008), but other studies report no difference (Fleg, O'Connor et al. 1995, Goldspink, George et al. 2009, Jakovljevic 2018). In addition to cardiac changes, the vasculature stiffens. Loss of blood vessel compliance occurs most prominently in the larger arteries which increases strain on the heart and contributes to systolic hypertension (Chirinos, Segers et al. 2019), a major risk factor for stroke in the elderly (Kocemba, Kawecka-Jaszcz et al. 1998). Furthermore, it contributes to the dysregulation of blood flow in response to local metabolic needs (i.e., reduced exercise-induced hyperemia in aged muscle (Proctor, Shen et al. 1998, Lawrenson, Poole et al. 2003)).

For basic pulmonary and cardiovascular research, rats are a common model for detailed oxygen supply and demand studies (Golub, Song et al. 2014, Schulze, Weber et al. 2022, Belbis, Yap et al. 2024, Hitosugi, Hotta et al. 2024, Song, Carr et al. 2024). They possess thin skeletal muscles, notably the dorsal spinotrapezius preparation described by Sarah D. Gray (Gray 1973), that lend themselves to brightfield microscopy and specific measurements of blood flow, vessel diameter, perfusion, oxygen delivery, oxygen uptake, and more. The majority of work in characterizing the rat oxygen transport chain (lungs, heart, vasculature, and mitochondrial utilization) has occurred in young rats approximating human early adulthood.

Aging studies in rats are challenged by the extensive resources needed to mature a colony. Aging studies are often performed in commercially available inbred or hybrid Fischer 344 rats (for relevant examples see refs (Behnke, Delp et al. 2005, Smith, Hageman et al. 2017, Horn, Schulze et al. 2024)), while outbred Sprague Dawley are common in physiological studies of young adulthood (Smith, Varagic et al. 2016, Ferguson, Harral et al. 2018, Golub, Dodhy et al. 2018, Hirai, Craig et al. 2019, Schulze, Weber et al. 2022, Weber, Schulze et al. 2022). Here we present the comparative aging physiology of male Sprague Dawley rats as an assessment of translatability. Common time points of 3, 6, 12, 18, and 24 months old (approximately 11, 18, 30, 45, and 60 in human years, respectively) (Sengupta 2013) under typical surgical and anesthetic preparation for cardiovascular and microcirculatory measurements are used.

Animals

The following protocol and experimental procedures—performed by Song Biotechnologies, LLC—were approved by the Thomas D Morris, Inc. Institutional Animal Care and Use Committee (Protocol # 20-007) and are consistent with the National Institutes of Health’s (NIH) guidelines for the humane treatment of laboratory animals. Study subjects were sixty-one (61) male Sprague Dawley rats (3-24 months old; purchased at 3-6 months of age from Charles River Laboratories, Inc., Wilmington, MA, USA). They were housed in pairs in independently ventilated cages (Innorack IVC with R-BTM-CH Innocages; Innovive, San Diego, CA) on a 12/12 day/night cycle and provided ad libitum access to feed (Teklad 2016 Global 16% Protein; Inotiv, West Lafayette, IN), water, and enrichment. All 12, 18, and 24 Month animals were aged in the Song Biotechnolgoies, LLC vivarium.

Surgical Preparation

Animals were weighed (Cole Palmer CS-200 scale; Antylia Scientific, Vernon Hills, IL) and inducted into anesthesia with 1 – 5% isoflurane in air for initial preoperative preparation and cannulations—right femoral vein and artery. The anesthetic plane was maintained with a continuous infusion of Alfaxalone acetate (IV; 0.1 mg/kg/min; Alfaxan, Schering-Plough Animal Health, Welwyn Garden City, UK) into the femoral vein using a syringe pump (Genie Touch™, Kent Scientific, Torrington, CT). The femoral artery cannula was connected to a pressure transducer for continuous monitoring of cardiovascular parameters (BIOPAC MP-160, BIOPAC Systems, Goleta, CA; Table 1). The same femoral artery cannula was also used to collect blood samples for analysis of blood gases and chemistry (ABL90 Flex, Radiometer, Denmark; Tables 2-4). All cannulas were kept free of clots with heparinized phosphate-buffered saline (20 IU Heparin Sodium per ml; Pfizer, New York, NY), which was not infused into the animal. A PE 240 tracheal tube was inserted to maintain airway patency, and animals continued to inspire room air without artificial ventilation.

The rat spinotrapezius muscle was utilized to investigate microcirculatory parameters and oxygen delivery. This thin skeletal muscle was exteriorized as described by Gray (Gray 1973) and secured in situ onto a custom, 3D-printed version of a thermostable animal platform adapted for microcirculatory studies (Golub and Pittman 2003, Song, Nugent et al. 2014, Nugent, Song et al. 2016). Animal core temperature was maintained at 37°C while the spinotrapezius prep on the thermostatic pedestal stayed at 36.5°C. A transparent sheet (~10x8 cm) of oxygen barrier film, Krehalon (CB-100; Krehalon Limited, Japan), was used to cover the preparation, isolate it from atmospheric contamination, and prevent desiccation. After the experiment, while under anesthesia, rats were euthanized with an IV infusion of Euthasol (150 mg/kg, pentobarbital component; Delmarva, Midlothian, Virginia).

Intravital Microscopy

Observation and measurements of the spinotrapezius microcirculation were carried out with an intravital microscope (Axioimager2m, Carl Zeiss, Jena, Germany) configured for trans-illumination through a 5X/0.25 objective (FLUAR, Zeiss). Trans-illumination was used to select measurement sites, establish appropriate focal planes, and verify flow conditions. The objective lens was focused in the diametral plane, and the image was displayed on a 4K resolution monitor.

Phosphorescence Quenching Microscopy

Measurements of the partial pressure of oxygen in interstitial fluid (P_ISFO₂) of the exteriorized spinotrapezius muscle and the technical setup have been previously described in depth (Nugent, Sheppard et al. 2019, Nugent, Carr et al. 2020, Nugent, Carr et al. 2021). Briefly, a phosphor (Oxyphore R0; Frontier Scientific, Newark, DE) bound to albumin (Bovine Serum Albumin, Sigma Aldrich, St. Louis, MO) was topically diffused into the spinotrapezius’ interstitium. The phosphor probe was excited at 1 Hz flash rate by a green laser diode operating at 520 nm (NDG7475 1W; Nichia Tokushima, Japan) in an octagonal region 300 µm in diameter. The excitation light pulse (1 µs duration) was reflected by a dichroic mirror (567nm DMLP Longpass, Thorlabs, Newton, NJ) to tissue. Emitted phosphorescence was collected by the objective lens and passed through a long-pass emission filter (Longpass Cut-on >650nm, Thorlabs, Newton, NJ). The phosphorescence signal was detected by a photomultiplier tube (R9110, Hamamatsu, Shizuoka, Japan), routed through a custom-built trans-impedance amplifier to a data acquisition board (NI PCIe-6361, National Instruments, Austin TX), and stored digitally on a computer. The resulting multi-exponential curves were processed using LabView-based code and the Levenberg-Marquart algorithm to fit the data employing a rectangular lifetime distribution model (Golub, Popel et al. 1997). The P_ISFO₂ values obtained every second were presented as a graph and statistical parameters of the time series.

Experimental Protocol

Following recovery from surgery, while remaining under anesthesia, measurements were recorded under “baseline” conditions. Animals were not treated or subjected to any experimental manipulation before or during baseline measurements. All animals were treated identically through the recording of baseline. All experiments were non-survival and ended in euthanasia with continuous anesthesia throughout.

Statistics

Data are expressed as mean ± standard error of the mean (SEM). Statistical comparisons were made between experimental groups with One-way ANOVA (Prism 9). Significance was defined as p < 0.05. A high stringency multiple comparison test (Tukey’s HSD or Sidak’s) was performed when a significant difference was detected.

Data Sharing:

For original data, please contact [email protected].

Systemic Physiological Parameters

Rats aged in independently ventilated microisolator cages with enrichment and socialization. No communicable diseases were detected among study animals, nor were they exposed to environmental toxins and pollutants that we are aware of. Air was filtered, and food and water were freely available and nutritionally balanced. Still, aging showed a progressive decline in physiology. Weight increased with age until 12 Months when it statistically plateaued. Intrinsic heart rates declined with age, significantly so from youth for 12, 18, and 24 Months. Mean arterial pressure dipped at 12 Months, but otherwise, it and pulse pressure did not trend or show differences with age. When blood pressure was aseparated into systolic and diastolic components, decreased diastole was detected at 24 Months (Table 1).

Metabolism

The fuel (oxygen and glucose), output (CO₂ and lactate), and byproducts (pH) of metabolism were assessed systemically with the ABL-90 arterial blood gas machine. Arterial oxygenation (p_AO₂), the driving force of oxygen delivery to tissues, declined with age. This was not associated with changes to aerobically-produced p_ACO₂, but a rise in anaerobic lactate for 12 and 18 Months. Blood glucose was elevated in 18 Months at 171±6 mg/dL, which was below the 200 mg/dL threshold for diabetes. The blood also became more alkaline in higher age groups (Table 2).

Co-oximetry

Overall, oxygen saturation, and non-functional forms of hemoglobin (metHb and COHb) were not sensitive to age. MetHb was elevated for 6 Months, but no additional trends or associations were noted. Total hemoglobin decreased by 2 g/dL by 24 Months (p=0.03 vs 3 Months; Table 3).

Electrolytes

Sodium, calcium, and chloride ions were conserved between groups. 24 Months showed a significant dip in potassium (versus 12 Months, p=0.032) and a rise in HCO₃^- (Table 4), which were consistent with rising blood alkalinity (Table 2).

Skeletal Muscle Oxygenation

Under anesthesia, the spinotrapezius muscle was at metabolic rest with a constant rate of oxygen consumption. Therefore, changes to P_ISFO₂—the balance between arteriolar supply and mitochondrial consumption—were indicative of oxygen delivery. Oxygenation of the spinotrapezius skeletal muscle tissue was within the normoxic range (>25 mmHg) for all groups, but a numeric decline was seen with increasing age, and a statistically significant drop from 62 mmHg in 18 Months to 43 mmHg in 24 Months was detected (p=0.02; Fig. 1). This resting state decline is consistent with the decreases noted above for P_AO₂.

Aging is a complex phenomenon that drives organisms to a universal endpoint but is often species-specific in its progression. Rodents are small laboratory animals with high metabolisms, short generation times, and lymphocyte-dominant blood immune profiles (Delwatta, Gunatilake et al. 2018) that are better tuned to the lifestyle of scavengers. Humans have slower metabolisms, neutrophil dominant blood counts, and are long-lived. Still, the cardiovascular systems of these two species are sufficiently conserved for translational research (Papadimitriou, Xanthos et al. 2008) and appear to senesce in relatively similar fashions.

This study confirms that rats, like humans, have declining heart rates as they age. Human data tends to report the age-associated decline in maximal heart rate during stress tests, but where there is a report of a 30% decline in intrinsic rates (Christou and Seals 2008), the same was found for rats (-29% calculated 3 vs. 24 months, Table 1) despite their 6–8 fold higher intrinsic rate. The cardiovascular element that appears to be lost is the age-related, intrinsic rises in systole, diastole, and MAP seen in humans (Franklin 1999, Kearney, Whelton et al. 2005). This may be due in part to the use of anesthesia for study rats versus conscious humans, but Alfaxan is minimally cardio-suppressive, and relative differences between groups are reasonably expected to remain intact, especially since anesthetized Sprague Dawley rats are often used in nitric oxide synthase-depleted hypertensive models. The lack of blood pressure change highlights the physical differences between rats and humans, where age-related isolated systolic hypertension is due to large artery stiffening. Rat aortas are essentially the same size as human intra-organ arterioles, which are part of the microcirculation. Direct comparative analysis and scaling are impossible when comparing human and rat aortas working at the same blood pressure. Understanding the structure of the aorta in both species is a separate and complex matter.

In humans, the decline in p_AO₂ without a corresponding decrease in p_ACO₂ is linked to age-increased pulmonary residual volume, decreased vital capacity, and decreased ventilatory response as discussed above (Sharma and Goodwin 2006). In aged rats, it might be related to findings of decreased resting oxygen consumption (McCarter, Herlihy et al. 1989). A main driver of senescence appears to be the degradation of the mitochondrial genome. Over time, ROS attacks and degrades mitochondrial DNA more rapidly than nuclear DNA due to its high replicative index, the limited efficiency of its repair mechanisms, its oxidative microenvironment, and the lack of protective histone (Lopez-Otin, Blasco et al. 2023). Inefficiency in mitochondrial replication and protein production then leads to inefficiency in function and therefore metabolism, which would explain why P_ISFO₂ and p_ACO₂ do not change despite reduced p_AO₂.

The developing hypoxemia in aging rats presents an interesting vascular mechanism. Declining central p_AO₂ without a corresponding decrease in oxygen delivery to the peripheral tissues or an increase in p_ACO₂ is consistent with humans. In humans, the phenomenon is explained by decreased ventilatory response to reduced p_AO₂ and p_ACO₂ (Sharma and Goodwin 2006), but not in rats (Wenninger, Olson et al. 2009). An alternative is the vascular response to hypoxemia, which usually shows peripheral constriction to preserve central blood flow and oxygenation. While arteriolar diameters were not measured, we consider the possibility of dysregulation of the peripheral circulation in response to hypoxemia. Already there is evidence for reduced hyperemia in rats (Hammer and Boegehold 2005) and humans (Citherlet, Raberin et al. 2024). Combined with evidence of declining carotid body function in humans (Di Giulio 2018) and rats (Conde, Obeso et al. 2006), the vasoconstrictive response to hypoxemia, often called “blood shunting” could also be compromised in both species. This would explain why oxygen delivery does not decline with hypoxemia, because blood flow is not being redirected away from the periphery to compensate. This mechanism is consistent with preliminary findings that older rats are less tolerant to blood loss, showing severe shock at 30% of total volume bleed whereas younger rats do not present similarly until 45% blood loss (data not published).

Despite the relatively short, 2–3 year, lifespan of rats, producing a suitably controlled, senescent colony is challenging. Many rat strains are prone to comorbidities that interfere with studies on healthy aging. In the 70s, the National Institute on Aging developed aging colonies for two species: Sprague Dawley and Fischer 344. The Sprague Dawley colony ended due to limited use and F344 (and hybridized variants) became the dominant strain for gerontological studies (Lipman, Chrisp et al. 1996). Since Sprague Dawley is extensively used in oxygen transport research, an area this longitudinal study shows is fundamental to aging, we provide baseline reference values of standard physiological parameters at commonly studied ages. The oxygen measurements made from the spinotrapezius preparation coupled with PQM are likely the greatest source of variation. Absolute tissue PO₂ values are subject to the style of tissue exteriorization (barrier film, water immersion, in situ), correct maintenance of tissue temperature (our thermostatic microscopy pedestal maintained 36.5°C), type and route of anesthesia, and the style and calculus of PQM—a vast area of discussion that transcends this paper. But the relative relationships of tissue PO₂, (measured here as P_ISFO₂), between groups should remain consistent across methodologies.

Limitations: Animals were anesthetized and surgically prepared for microcirculatory studies, which has an impact on the absolute cardiovascular-dependent values. This study also only evaluated males. There are well-described differences in cardiovascular function between sexes and female aging studies remain lacking. Our goal was to isolate age-related changes while navigating the challenges of raising a senescent colony. Males were chosen due to their existing predominance in referenceable literature and more consistent endocrinological function. We believe, however, that while absolute values for the metrics presented may vary for females, the relative changes with advancing age should remain consistent and an interesting area for study and development of testable models.

The study's objective was to conduct a longitudinal analysis of the fundamental physiological characteristics of laboratory Sprague Dowley rats to determine consistent parameters and those that vary with age. The primary findings are that cardiac, oxygen delivery and microvascular function are reasonably conserved with humans, offering testable models for declining heart rates, and age-related hypoxemia and hypercapnia. Paradoxical to declining cardiovascular function was unchanged oxygen delivery to the peripheral skeletal muscle tissue which is typically reduced under the observed conditions of hypoxemia. It may be indicative of blood shunting dysregulation with implications for increased mortality related to hypoxia in the elderly.

Authorship contributions

William Nugent: Study design, data collection, data analysis, writing.

Bjorn Song: Study design, data collection, writing.

Aleksander Golub: Study design, data collection, writing

Roland Pittman: Study design, writing

Disclosure of Interest

The authors have no conflicts to declare.

Funding

Study was funded by Song Biotechnologies, LLC.

Study and manuscript were funded by Song Biotechnologies, LLC.

Author Contribution

W.H.N.: Study design, data collection, data analysis, writing.B.K.S.: Study design, data collection, writing.A.S.G.: Study design, data collection, writingR.N.P.: Study design, writing

Acknowledgments

None

Data Availability

Summarized data (mean +- SEM) are provided in the manuscript. For additional information, contact [email protected].

Amorim, J. A., G. Coppotelli, A. P. Rolo, C. M. Palmeira, J. M. Ross and D. A. Sinclair (2022). "Mitochondrial and metabolic dysfunction in ageing and age-related diseases." Nat Rev Endocrinol 18(4): 243–258.
Behnke, B. J., M. D. Delp, P. J. Dougherty, T. I. Musch and D. C. Poole (2005). "Effects of aging on microvascular oxygen pressures in rat skeletal muscle." Respir Physiol Neurobiol 146(2–3): 259–268.
Belbis, M. D., Z. Yap, S. E. Hobart, S. K. Ferguson and D. M. Hirai (2024). "Effects of acute phosphodiesterase type 5 inhibition on skeletal muscle interstitial PO(2) during contractions and recovery." Nitric Oxide 142: 16–25.
Brischetto, M. J., R. P. Millman, D. D. Peterson, D. A. Silage and A. I. Pack (1984). "Effect of aging on ventilatory response to exercise and CO2." J Appl Physiol Respir Environ Exerc Physiol 56(5): 1143–1150.
Brooks, S. V. and J. A. Faulkner (1994). "Skeletal muscle weakness in old age: underlying mechanisms." Med Sci Sports Exerc 26(4): 432–439.
Cerveri, I., M. C. Zoia, F. Fanfulla, L. Spagnolatti, L. Berrayah, M. Grassi and C. Tinelli (1995). "Reference values of arterial oxygen tension in the middle-aged and elderly." Am J Respir Crit Care Med 152(3): 934–941.
Chapman, K. R. and N. S. Cherniack (1987). "Aging effects on the interaction of hypercapnia and hypoxia as ventilatory stimuli." J Gerontol 42(2): 202–209.
Chirinos, J. A., P. Segers, T. Hughes and R. Townsend (2019). "Large-Artery Stiffness in Health and Disease: JACC State-of-the-Art Review." J Am Coll Cardiol 74(9): 1237–1263.
Christou, D. D. and D. R. Seals (2008). "Decreased maximal heart rate with aging is related to reduced beta-adrenergic responsiveness but is largely explained by a reduction in intrinsic heart rate." J Appl Physiol (1985) 105(1): 24–29.
Citherlet, T., A. Raberin, G. Manferdelli, G. R. Mota and G. P. Millet (2024). "Age and sex differences in microvascular responses during reactive hyperaemia." Exp Physiol 109(5): 804–811.
Conde, S. V., A. Obeso, R. Rigual, E. C. Monteiro and C. Gonzalez (2006). "Function of the rat carotid body chemoreceptors in ageing." J Neurochem 99(3): 711–723.
Delwatta, S. L., M. Gunatilake, V. Baumans, M. D. Seneviratne, M. L. B. Dissanayaka, S. S. Batagoda, A. H. Udagedara and P. B. Walpola (2018). "Reference values for selected hematological, biochemical and physiological parameters of Sprague-Dawley rats at the Animal House, Faculty of Medicine, University of Colombo, Sri Lanka." Animal Model Exp Med 1(4): 250–254.
Di Giulio, C. (2018). "Ageing of the carotid body." J Physiol 596(15): 3021–3027.
Ferguson, S. K., J. W. Harral, D. I. Pak, K. M. Redinius, K. R. Stenmark, D. J. Schaer, P. W. Buehler and D. C. Irwin (2018). "Impact of cell-free hemoglobin on contracting skeletal muscle microvascular oxygen pressure dynamics." Nitric Oxide 76: 29–36.
Fleg, J. L., F. O'Connor, G. Gerstenblith, L. C. Becker, J. Clulow, S. P. Schulman and E. G. Lakatta (1995). "Impact of age on the cardiovascular response to dynamic upright exercise in healthy men and women." J Appl Physiol (1985) 78(3): 890–900.
Franklin, S. S. (1999). "Cardiovascular risks related to increased diastolic, systolic and pulse pressure. An epidemiologist's point of view." Pathol Biol (Paris) 47(6): 594–603.
Goldspink, D. F., K. P. George, P. D. Chantler, R. E. Clements, L. Sharp, G. Hodges, C. Stephenson, T. P. Reilly, A. Patwala, T. Szakmany, L. B. Tan and N. T. Cable (2009). "A study of presbycardia, with gender differences favoring ageing women." Int J Cardiol 137(3): 236–245.
Golub, A. S., S. C. Dodhy and R. N. Pittman (2018). "Oxygen dependence of respiration in rat spinotrapezius muscle contracting at 0.5-8 twitches per second." J Appl Physiol (1985) 125(1): 124–133.
Golub, A. S. and R. N. Pittman (2003). "Thermostatic animal platform for intravital microscopy of thin tissues." Microvasc Res 66(3): 213–217.
Golub, A. S., A. S. Popel, L. Zheng and R. N. Pittman (1997). "Analysis of phosphorescence in heterogeneous systems using distributions of quencher concentration." Biophys J 73(1): 452–465.
Golub, A. S., B. K. Song and R. N. Pittman (2014). "Muscle contraction increases interstitial nitric oxide as predicted by a new model of local blood flow regulation." J Physiol 592(6): 1225–1235.
Gray, S. D. (1973). "Rat spinotrapezius muscle preparation for microscopic observation of the terminal vascular bed." Microvasc Res 5(3): 395–400.
Hammer, L. W. and M. A. Boegehold (2005). "Functional hyperemia is reduced in skeletal muscle of aged rats." Microcirculation 12(6): 517–526.
Hirai, D. M., J. C. Craig, T. D. Colburn, H. Eshima, Y. Kano, T. I. Musch and D. C. Poole (2019). "Skeletal muscle interstitial Po2 kinetics during recovery from contractions." Journal of Applied Physiology 127(4): 930–939.
Hitosugi, N., K. Hotta, Y. Taketa, R. Takamizawa, Y. Fujii, R. Ikegami, H. Tamiya, T. Inoue and A. Tsubaki (2024). "The effect of sepsis and reactive oxygen species on skeletal muscle interstitial oxygen pressure during contractions." Microcirculation 31(1): e12833.
Hood, D. A., J. M. Memme, A. N. Oliveira and M. Triolo (2019). "Maintenance of Skeletal Muscle Mitochondria in Health, Exercise, and Aging." Annu Rev Physiol 81: 19–41.
Horn, A. G., K. M. Schulze, J. Muller-Delp, D. C. Poole and B. J. Behnke (2024). "Effects of aging on diaphragm hyperemia and blood flow distribution in male and female Fischer 344 rats." Am J Physiol Regul Integr Comp Physiol 327(3): R328-R337.
Jakovljevic, D. G. (2018). "Physical activity and cardiovascular aging: Physiological and molecular insights." Exp Gerontol 109: 67–74.
Kearney, P. M., M. Whelton, K. Reynolds, P. Muntner, P. K. Whelton and J. He (2005). "Global burden of hypertension: analysis of worldwide data." Lancet 365(9455): 217–223.
Kocemba, J., K. Kawecka-Jaszcz, B. Gryglewska and T. Grodzicki (1998). "Isolated systolic hypertension: pathophysiology, consequences and therapeutic benefits." J Hum Hypertens 12(9): 621–626.
Lawrenson, L., J. G. Poole, J. Kim, C. Brown, P. Patel and R. S. Richardson (2003). "Vascular and metabolic response to isolated small muscle mass exercise: effect of age." Am J Physiol Heart Circ Physiol 285(3): H1023-1031.
Lipman, R. D., C. E. Chrisp, D. G. Hazzard and R. T. Bronson (1996). "Pathologic characterization of brown Norway, brown Norway x Fischer 344, and Fischer 344 x brown Norway rats with relation to age." J Gerontol A Biol Sci Med Sci 51(1): B54-59.
Lopez-Otin, C., M. A. Blasco, L. Partridge, M. Serrano and G. Kroemer (2023). "Hallmarks of aging: An expanding universe." Cell 186(2): 243–278.
McCarter, R. J., J. T. Herlihy and J. R. McGee (1989). "Metabolic rate and aging: effects of food restriction and thyroid hormone on minimal oxygen consumption in rats." Aging (Milano) 1(1): 71–76.
Nugent, W. H., D. A. Carr, R. MacBryde, E. D. Bruce and B. K. Song (2021). "Gavage approach to oxygen supplementation with oxygen therapeutic Ox66 in a hypoventilation rodent model of respiratory distress." Artif Cells Nanomed Biotechnol 49(1): 709–716.
Nugent, W. H., D. A. Carr, A. R. Macko and B. K. Song (2020). "Physiological and microvascular responses to hemoglobin concentration-targeted hemolytic anemia in rats." J Appl Physiol (1985) 128(6): 1579–1586.
Nugent, W. H., F. R. Sheppard, M. A. Dubick, R. F. Cestero, D. N. Darlington, R. Jubin, A. Abuchowski and B. K. Song (2019). "Microvascular and Systemic Impact of Resuscitation with Pegylated Carboxyhemoglobin-Based Oxygen Carrier or Hetastarch In A Rat Model of transient hemorrhagic Shock." Shock.
Nugent, W. H., B. K. Song, R. N. Pittman and A. S. Golub (2016). "Simultaneous sampling of tissue oxygenation and oxygen consumption in skeletal muscle." Microvasc Res 105: 15–22.
Papadimitriou, D., T. Xanthos, I. Dontas, P. Lelovas and D. Perrea (2008). "The use of mice and rats as animal models for cardiopulmonary resuscitation research." Lab Anim 42(3): 265–276.
Peters, C. H., E. J. Sharpe and C. Proenza (2020). "Cardiac Pacemaker Activity and Aging." Annu Rev Physiol 82: 21–43.
Peterson, D. D., A. I. Pack, D. A. Silage and A. P. Fishman (1981). "Effects of aging on ventilatory and occlusion pressure responses to hypoxia and hypercapnia." Am Rev Respir Dis 124(4): 387–391.
Proctor, D. N., P. H. Shen, N. M. Dietz, T. J. Eickhoff, L. A. Lawler, E. J. Ebersold, D. L. Loeffler and M. J. Joyner (1998). "Reduced leg blood flow during dynamic exercise in older endurance-trained men." J Appl Physiol (1985) 85(1): 68–75.
Rana, J. S., S. S. Khan, D. M. Lloyd-Jones and S. Sidney (2021). "Changes in Mortality in Top 10 Causes of Death from 2011 to 2018." J Gen Intern Med 36(8): 2517–2518.
Schulze, K. M., R. E. Weber, A. G. Horn, T. D. Colburn, C. J. Ade, D. C. Poole and T. I. Musch (2022). "Effects of pulmonary hypertension on microcirculatory hemodynamics in rat skeletal muscle." Microvasc Res 141: 104334.
Sengupta, P. (2013). "The Laboratory Rat: Relating Its Age With Human's." Int J Prev Med 4(6): 624–630.
Sharma, G. and J. Goodwin (2006). "Effect of aging on respiratory system physiology and immunology." Clin Interv Aging 1(3): 253–260.
Smith, J. R., K. S. Hageman, C. A. Harms, D. C. Poole and T. I. Musch (2017). "Effect of chronic heart failure in older rats on respiratory muscle and hindlimb blood flow during submaximal exercise." Respir Physiol Neurobiol 243: 20–26.
Smith, L. M., J. Varagic and L. M. Yamaleyeva (2016). "Photoacoustic Imaging for the Detection of Hypoxia in the Rat Femoral Artery and Skeletal Muscle Microcirculation." Shock 46(5): 527–530.
Song, B. K., D. A. Carr, E. D. Bruce and W. H. Nugent (2024). "Oxygenation through oral Ox66 in a two-hit rodent model of respiratory distress." Artif Cells Nanomed Biotechnol 52(1): 114–121.
Song, B. K., W. H. Nugent, P. F. Moon-Massat and R. N. Pittman (2014). "Effects of a hemoglobin-based oxygen carrier (HBOC-201) and derivatives with altered oxygen affinity and viscosity on systemic and microcirculatory variables in a top-load rat model." Microvasc Res 95: 124–130.
Weber, R. E., K. M. Schulze, T. D. Colburn, A. G. Horn, K. S. Hageman, C. J. Ade, S. E. Hall, P. Sandner, T. I. Musch and D. C. Poole (2022). "Capillary hemodynamics and contracting skeletal muscle oxygen pressures in male rats with heart failure: Impact of soluble guanylyl cyclase activator." Nitric Oxide 119: 1–8.
Wenninger, J. M., E. B. Olson, Jr., C. J. Cotter, C. F. Thomas and M. Behan (2009). "Hypoxic and hypercapnic ventilatory responses in aging male vs. aging female rats." J Appl Physiol (1985) 106(5): 1522–1528.

Table 1: Systemic Physiological Parameters

Age	Weight	MAP	Systolic	Diastolic	HR	PP	N
Months	grams	mmHg	mmHg	mmHg	BPM	mmHg
3	584±19	128±4	151±5	117±4	417±9	34±2	11
6	680±28	107±8	128±8	97±7	410±11	31±2	10
12	813±34^α	101±5^α	125±6	92±5	341±11^α,β	35±3	16
18	814±45^α	105±7	127±9	93±7	362±10^α	37±5	13
24	924±35^α,β	105±6	128±7	87±7^α	314±16^α,β	42±3	14

Table 1: Systemic. Measurements were performed on anesthetized Sprague Dawley rats following surgical cannulation and exteriorization of the spinotrapezius muscle for microscopy. These conditions conform to typical baseline measurements for non-survival studies. Measurements will vary per anesthetic protocol (e.g., IM ketamine versus inhaled isoflurane) but relative relationships between age groups should remain. Data are mean ± SEM.

MAP: Mean Arterial Pressure, HR: Heart Rate, PP: Pulse Pressure

^α p < 0.05 versus 3 Months

^β p < 0.05 versus 6 Months

Table 2: Metabolism

Age	p_AO₂	p_ACO₂	Glucose	Lactate	pH	Base Excess	N
Months	mmHg	mmHg	mg/dL	mg/dL	-log[H+]	mmol/L
3	91.9±2.4	39.4±0.9	144±6	10±1	7.34±0.01	-4.5±0.9	11
6	88.4±3.0	36.6±1.7	119±7	9±1	7.38±0.01^α	-3.4±0.8	10
12	84.6±2.4	38.5±1.0	138±11	16±2 ^α,β	7.38±0.01^α	-2.2±0.5	13
18	79.0±2.1^α	36.4±0.8	171±6 ^β	18±1^α,β	7.40±0.01^α	-2.4±0.6	13
24	81.3±2.3^α	38.3±1.2	133±15	14±1	7.41±0.01^α	-0.7±0.9^α	12

Table 2: Metabolism. These metabolic indicators are related to circulatory gas transport and aerobic and anaerobic metabolism. Measurements were taken from arterial blood in anesthetized rats. Data are mean ± SEM.

p_AO₂: Partial Pressure of Oxygen in Arterial Blood, p_ACO₂: Partial Pressure of CO₂ in Arterial Blood

^α p < 0.05 versus 3 Months

^β p < 0.05 versus 6 Months

Table 3: Co-oximetry

Age	tHb	S_AO₂	O₂Hb	COHb	HHb	MetHb	N
Months	g/dL	%	%	%	%	%
3	16.3±0.3	92±1	92±1	0.0±0	8.0±.5	0.1±0	11
6	15.8±0.6	94±1	93±1	0.7±0.3	5.5±1.0	0.5±0.1^α	10
12	15.3±0.4	92±1	92±1	0.3±0.2	7.7±1.0	0.3±0.1	13
18	15.4±0.5	92±1	91±1	0.2±0.1	8.3±1.4	0.3±0.1	13
24	14.4±0.3^α	92±1	91±1	0.2±0.2	8.0±1.0	0.4±0.1	12

tHb: Total Hemoglobin, Arterial Oxygen Saturation: O₂Hb: Oxyhemoglobin, COHb: Carboxyhemoglobin: HHb: Deoxyhemoglobin, MetHb: Methemoglobin

Table 3: Co-oximetry. Measurements were performed on arterial blood from anesthetized rats. Animals were breathing room air with spontaneous respiration through a PE240 tracheal tube at a length that maintained normal pulmonary dead space. Data are mean ± SEM.

^α p < 0.05 versus 3 Months

Table 4: Electrolytes

Age	K⁺	Na⁺	Ca²⁺	Cl^-	HCO₃^-	N
Months	meq/L	meq/L	meq/L	meq/L	mmol/L
3	3.6±0.1	147±1	1.7±0.1	113±1	21±0.5	11
6	4.0±0.1	146±1	1.7±0.1	111±2	22±0.5	10
12	4.0±0.1	144±2	1.7±0.1	108±1	23±0.3	13
18	3.8±0.1	145±1	1.7±0.0	110±1	22±0.4	13
24	3.4±0.1^β	146±1	1.7±0.1	110±1	24±0.6^α	12

Table 4: Electrolytes. Ions were measured from arterial blood samples. Concentrations are generally expected to be consistent unless other pathologies are present. The minor shifts seen with the 24 Month group are believed to be age-related, but not outside the normal ranges for K⁺ and HCO₃^-. Data are mean ± SEM.

K+: Potassium Cation, Na+: Sodium Cation: Ca²⁺: Calcium Cation, Cl^-: Chloride Anion: HCO₃^-: Bicarbonate Anion

^α p < 0.05 versus 3 Months

^β p < 0.05 versus 6 Months

No competing interests reported.

Baseline Cardiovascular and Oximetric Parameters Across the Lifespan of Male Sprague Dawley Rats

Status:

Version 1

Abstract

Figures

Introduction

Methods

Results

Systemic Physiological Parameters

Metabolism

Co-oximetry

Electrolytes

Skeletal Muscle Oxygenation

Discussion

Conclusion

Declarations

Authorship contributions

Disclosure of Interest

Funding

Author Contribution

Acknowledgments

Data Availability

References

Tables

Table 1: Systemic Physiological Parameters

Table 2: Metabolism

Table 3: Co-oximetry

Table 4: Electrolytes

Additional Declarations

Status:

Version 1