Tail Tendon Break Time for the Assessment of Aging and Longitudinal Healthspan in Mice

Background

The field of gerontology research and the identification of geroprotectors in the form of pharmacological interventions require reliable methods for quantifying an animal’s biological age and assessing their health status during aging (Sprott, 2010; Kennedy et al., 2014; Richardson et al., 2016; de Magalhaes et al., 2017; Bellantuono, 2018; Bellantuono et al., 2020). The availability of a biological marker, defined as a characteristic that can be measured objectively and that can differentiate between the normal biological process of aging and altered aging processes due to interventions would be of major benefit to geroscience (Biomarkers Definitions Working Group, 2001; Thompson and Voss, 2009). To date, commonly used biomarkers have often required comparing the lifespan and healthspan of the animals. Unfortunately, however, studies that directly require measurements of the lifespan of a mouse model require two to four years to complete. Therefore, the identification of a biomarker capable of reflecting the differences in biological aging processes at earlier stages remains critical (Levine et al., 2018). Here, we discuss the measurement of Tail Tendon Break Time (TTBT) as an indirect readout for age-related collagen crosslinking. We find that while TTBT might not fulfill all criteria as a bona fide biomarker of aging, TTBT remains a valuable indicator of the murine aging process during longitudinal healthspan assessments.

Collagens are structural proteins produced and secreted by cells to form scaffolds in the extracellular matrix. Collagens can be long-lived structural proteins, especially in acellular tissues; for example, collagens have been found to have a half-life time of more than one hundred years in human eye lenses (Birch, 2018; Ewald, 2019). The general decline of protein turnover during aging leads to the accumulation of non-enzymatic glycation on proteins, including collagens, due to a phenomenon called the Maillard reaction (Avery and Bailey, 2006). In this process, non-enzymatic glycation results in an increase in non-enzymatic crosslinking of collagen (Avery and Bailey, 2005; Snedeker and Gautieri, 2014) and the addition of side-chain modifications. This process is accelerated under increased temperatures and under conditions in which there is higher availability of glucose. The adducts and crosslinking of Advanced Glycation End products (AGEs) on collagens during aging cause significant changes in physical properties of collagen fibers, such as increased tissue bending stiffness, decreased tissue viscoelasticity, and reduced susceptibility towards denaturing agents (Avery and Bailey, 2006; Snedeker and Gautieri, 2014; Gautieri et al., 2017). The increased resistance of collagen fibers to denaturing agents can be directly linked to biological aging (Paul and Bailey, 1996). Thus, the TTBT assay, which measures the resistance of a single tail tendon fascicle to elongation under the creep load of a single tail tendon fascicle during the denaturation of the fiber by concentrated urea, provides an indirect measure of the amount of collagen crosslinking (Harrison and Archer, 1978). The measurement of TTBT can be performed at least four times in an animal’s lifetime by using each of the animal’s four individual tendon bundles once. Tendon fascicles can be harvested from living animals using local anesthesia, which offers the possibility for longitudinal sampling (Harrison and Archer, 1978). Furthermore, a mouse’s tail can be stored at -20 °C without influencing the mechanical properties of its collagen fibers (Goh et al., 2008).

For almost seventy years, TTBT assays have been employed to assess tendon stiffness during the aging process (Verzar, 1955). Numerous studies have reported that older animals exhibit increased TTBTs when compared to younger animals of the same sex and strain (Olsen and Everitt, 1965; Harrison and Archer, 1978; Sell and Monnier, 1997). A comparison between the longer-lived mouse species Peromyscus leucopus with the shorter-lived species Mus musculus, for example, reveals a more rapid increase in TTBT in the shorter-lived species across age (Harrison et al., 1978). Furthermore, short-lived mouse strains exhibit longer TTBTs than long-lived strains at the same chronological age (Harrison et al., 1984; Higgins et al., 1991; Heller and McClearn, 1992; Sloane et al., 2011a and 2011b). Thus, a positive relationship between age and tail tendon breakage time has been found consistently across studies in rodents.

If non-enzymatic AGE-crosslinking of aging collagens is an age-related pathology, then interventions that slow aging should reverse or prevent collagen crosslinking. In keeping with that hypothesis, by 1965, it had been found that hypophysectomized rats had both delayed signs of aging and a shortened TTBT comparable to about half of the chronological age of the control group (Olsen and Everitt, 1965). Moreover, interventions like caloric restriction, which are known to increase an animal’s lifespan, resulting in a shortened TTBT when compared to ad libitum fed animals from the same strain (Harrison et al., 1984; Sell and Monnier, 1997). In contrast, short-lived obese mice exhibit an increased TTBT compared to nonobese controls (Harrison et al., 1984). Similarly, a more youthful or decreased TTBT is found in long-lived Snell dwarf mice when compared to their shorter-lived sibling controls (Flurkey et al., 2001). TTBT assays have also been used to demonstrate the effect of lifespan-altering interventions involving hypothalamic programming, in which shorter-lived MBH-IKK-β mice exhibited greater TTBTs than both the control group and the longer-lived MBH-IκB-α mice (Zhang et al., 2013). Taken together, interventions that slow aging and increase lifespan slow collagen aging, as indicated by a more youthful TTBT.

Given that TTBT increases with age and this increase is slowed by longevity interventions, TTBT values have been considered as a biomarker of aging. The TTBT meets eight of the nine criteria for a biomarker of aging, including being minimally invasive, insensitive to anesthesia, and highly reproducible. TTBTs increase substantially with an animal’s age, and the age-related changes in TTBT can be slowed or reversed by longevity interventions.

Some caveats to the use of TTBT as a biomarker of aging remain, however. For example, TTBT values show minimal predictive power for predicting the length of the animals’ lifespan. Using 23 recombinant inbred strains (C57BL/6J and DBA/2J, i.e., BxD RIs), Sloane and colleagues have found that neither the rate of change in TTBT nor absolute TTBT values correlate significantly with the rate of increase in lifespan of BxD RIs (Sloane et al., 2011a). As no robust univariate statistical relationship between TTBT and lifespan was found, TTBT might not be suitable for direct correlations between chronological age and TTBT values (Sloane et al., 2011a). Moreover, many of the interventions that lower TTBT also lower glucose levels and temperature (Sloane et al., 2011a), which may directly lower the accumulation of AGEs and crosslinking of collagens, and therefore lead to a lower TTBT independent from their effects on aging. Finally, it remains unclear whether AGEs on collagens are actively prevented under conditions in which lifespan is prevented, or whether they are actively removed by these interventions. Nevertheless, we argue that the sensitivity of TTBT to aging as well as to longevity interventions makes it an important factor in the assessment of qualitative, age-related changes, even though TTBT might not be suitable as a bona fide biomarker of aging (Sloane et al., 2011a).

In this work, we review the knowledge gained by TTBT assays across the past 70 years. We discuss the advantages and potential short-comings of TTBT and its suitability as an assay to the aging process. We find that while TTBT is an established assay for the study of age-related changes to collagens and extracellular matrix, TTBT should be used in combination with additional assays, such as collagen deposition during aging (Teuscher et al., 2019). TTBT should be seen as a qualitative assessment of age-related changes. We therefore advise use the TTBT in combination with other longitudinal healthspan assays, as described by Bellantuono and colleagues (Bellantuono et al., 2020) when assessing healthspans.

General technical considerations for TTBT
For a general design of a study for the longitudinal assessment of health and lifespan that compares a longevity-promoting intervention (i.e., geroprotector) to a control, please see Bellantuono and colleagues (Bellantuono et al., 2020). When crafting a study’s design for longitudinal sampling, keep in mind the requirement of anesthesia and the limited number of times TTBT can be performed. The time brackets to measure significant changes in TTBT show different ranges in different mouse species (Harrison et al., 1984; Higgins et al., 1991; Heller and McClearn, 1992; Sloane et al., 2011a and 2011b). In general, for C57BL6 mice of the ages of 2 to 5 and 5 to 10 months, the TTBT is about double the time for each of these mouse age brackets (Higgins et al., 1991). From 6 to 17 and 17 to 27 months, the TTBT is about double or triple in time, respectively (Sloane et al., 2011b). For other mouse strains, such as DW/J or DBA, the TTBT measured at 6 and 18 months of age can increase by about 6 or 18-fold, respectively (Flurkey et al., 2001; Sloane et al., 2011b). Furthermore, fascicle sampling might impact other healthspan assays, such as rotarod performance or the Howlett/Rockwood frailty assay, for which the tail helps with balancing or assess tail curling, respectively. Caution in the preparation and execution of measuring TTBT is advised due to the following critical issues:

1. The non-enzymatic glycation reaction depends on the amount of circulating sugar and the temperature of the tissue (Harrison and Archer, 1978). Thus, researchers need to consider whether their intervention of interest will affect blood glucose levels. Furthermore, the body (rectal) temperature of mice is about 6 °C warmer than at the base of the tail and 9 °C warmer than the middle part of the tail. Due to this temperature difference, it is recommended to use fascicles from the middle part of the tail (Harrison and Archer, 1978). In addition, even subtle microenvironmental temperature differences create significant variations, depending on whether dorsal or ventral fascicles were used (Higgins et al., 1991). To minimize individual variation, housing temperature and the location of fascicles harvested from the tail should be kept constant. The difference in age-related increases in TTBT is usually far greater than these individual TTBT differences, however.
   

2. TTBT depends on the rate of penetration of the urea solution into the fascicle, which is proportional to the surface area per volume ratio (Harrison and Archer, 1978). Thus, a split fascicle will break faster, but a double fascicle will break similar to a single fascicle (Harrison and Archer, 1978).
   

3. TTBT depends on the urea solution temperature. The lower the temperature of the urea solution, the longer the TTBT. Around 45 °C is recommended for the solution (Harrison and Archer, 1978).
   

4. TTBT is not altered by anesthesia, fascicle length, dryness, or constant urea concentration (Harrison and Archer, 1978). The latter point is important since water will evaporate from the urea solution, and water needs to be re-added during longer measurements.