How (Not) to Run a Lifespan Study

Posted by Michael Rae on March 26, 2011 | Chief Science Officer's Team

Much of the distraction in the literature of biogerontology, and an even higher ratio of studies cited and promoted in the popular media and the dietary supplement industry, derives from methodologically-poor lifespan studies in mice (or occasionally rats). The pineal hormone melatonin,(1) the wine polyphenol resveratrol,(2) branched-chain amino acids,(3) the mitochondrially-targeted antioxidant plastoquinonyl decyltriphenyl phosphonium (SkQ1),(4,5) mice with a Fat-specific Insulin Receptor Knockout (FIRKO)(6), ribonucleic acid supplementation,(7) various vitamin mixtures,((8), misinterpretation of (11)'s honest reporting) the selective irreversible MAO-B inhibitor and Parkinson's disease treatment selegiline (L-deprenyl, Eldepryl)(9,10) ... a long series of instantiations of the Original Sin of biogerontology.

In these studies, an increase in mean or maximal lifespan is reported, relative to short-lived controls, and claimed to be informative about the universal, degenerative aging process and the prospects for extending healthy life in humans living in the developed world. And the claim typically persists for decades once widely-cited, despite the best efforts of serious investigators to critique weak methodologies and flawed interpretation (eg. (12,13)), or even robust demonstrations of a null effect in healthy animals (as the two independent demonstrations that resveratrol does not extend lifespan in nonobese, wild-type mice over a wide range of doses).

Extensive studies by careful investigators such as Dr. Stephen Spindler of UC Riverside, Dr. Richard Weindruch of the University of Wisconsin at Madison, and Dr. Richard Miller of the University of Michican have shown that careful husbandry of a healthy cohort of wild-type laboratory mice fed a nonobesogenic environment and maintained in specific pathogen-free laboratores will, on average, live to be  ~900 days, with a cohort maximum lifespan (operationally defined as tenth-decile survivorship) of ~1100 d. But in report after report of 'life extension' in mice, either none of the animals -- controls or intervention animals -- reach even this threshold lifespan, or else only the intervention group does -- and this result is declared to be a significant advance in our understanding of the aging process and its modulation.

The aforementioned Dr. Spindler has done a great deal of important Calorie restriction (CR) research, including the study definitively establishing that CR continues to be effective in early seniority (19-20 mo old at initiation),(14) and is one of the few investigators to run genuinely rigorous mouse lifespan studies, thereby debunking multitudes of purported "anti-aging" dietary supplements (eg., (15)).  Thus, the legitimate biogerontological community, and advocates of consumer protection, already owe a debt of gratitude to Spindler for his years of painstaking labor on our behalf in the laboratory.

Now,  Spindler has written an extensive review of the many flaws that litter the litters in the literature, and proposed concrete methods to avoid these flaws and confounds:

Much of the literature describing the search for agents that increase the life span of rodents was found to suffer from confounds. One-hundred-six studies, absent 20 contradictory melatonin studies, of compounds or combinations of compounds were reviewed.

Only six studies reported both life span extension and food consumption data, thereby excluding the potential effects of caloric restriction. Six other studies reported life span extension without a change in body weight. However, weight can be an unreliable surrogate measure of caloric consumption. Twenty studies reported that food consumption or weight was unchanged, but it was unclear whether these data were anecdotal or systematic.

Twenty-nine reported extended life span likely due to induced caloric restriction. Thirty-six studies reported no effect on life span, and three a decrease. The remaining studies suffer from more serious confounds.

Though still widely cited, studies showing life span extension using short-lived or "enfeebled" rodents have not been shown to predict longevity effects in long-lived animals.

We suggest improvements in experimental design that will enhance the reliability of the rodent life span literature. First, animals should receive measured quantities of food and its consumption monitored, preferably daily, and reported. Weights should be measured regularly and reported. Second, a genetically heterogeneous, long-lived rodent should be utilized. Third, chemically defined diets should be used. Fourth, a positive control (e.g., a calorically restricted group) is highly desirable. Fifth, drug dosages should be chosen based on surrogate endpoints or accepted cross-species scaling factors. These procedures should improve the reliability of the scientific literature and accelerate the identification of longevity and health span-enhancing agents.(16)

I was surprised to see that the state of the literature is even worse than I had realized, as  subtle flaws in execution and reporting that I'd've blithely passed over or accepted prove to be important to certainty about the meaning of the results, and bring into question several good-looking studies (although in most cases, reports with subtle flaws also have larger ones).

Happily, the full text of this comprehensive review is available online from the publisher -- a guide to designing, executing, and interpreting reports of rodent lifespan studies , by someone who knows from experience what is required to generate robust experimental results. There is much to gain from Spindler's years of experience, for investigators preparing to execute rodent longevity studies and for those seeking to understand the ensuing reports, for potential funders of such interventions, and for anyone wishing to become an informed reader of the literature on which progress toward an extension of healthy human lifespan may depend. This review should be considered required reading for those interested in advancing the science of aging rather than generating headlines or supplement sales.

References

1. Pierpaoli W, Dall'Ara A, Pedrinis E, Regelson W. The pineal control of aging. The effects of melatonin and pineal grafting on the survival of older mice. Ann N Y Acad Sci. 1991;621:291-313. PMID: 1859093 [PubMed - indexed for MEDLINE]

2: Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS, Lopez-Lluch G, Lewis K, Pistell PJ, Poosala S, Becker KG, Boss O, Gwinn D, Wang M, Ramaswamy S, Fishbein KW, Spencer RG, Lakatta EG, Le Couteur D, Shaw RJ, Navas P, Puigserver P, Ingram DK, de Cabo R, Sinclair DA. Resveratrol improves health and survival of mice on a high-calorie diet. Nature. 2006 Nov 16;444(7117):337-42. Epub 2006 Nov 1. PMID: 17086191 [PubMed - indexed for MEDLINE]

3: D'Antona G, Ragni M, Cardile A, Tedesco L, Dossena M, Bruttini F, Caliaro F, Corsetti G, Bottinelli R, Carruba MO, Valerio A, Nisoli E. Branched-chain amino acid supplementation promotes survival and supports cardiac and skeletal muscle mitochondrial biogenesis in middle-aged mice. Cell Metab. 2010 Oct 6;12(4):362-72. PubMed PMID: 20889128.

4: Skulachev VP, Anisimov VN, Antonenko YN, Bakeeva LE, Chernyak BV, Erichev VP, Filenko OF, Kalinina NI, Kapelko VI, Kolosova NG, Kopnin BP, Korshunova GA, Lichinitser MR, Obukhova LA, Pasyukova EG, Pisarenko OI, Roginsky VA, Ruuge EK, Senin II, Severina II, Skulachev MV, Spivak IM, Tashlitsky VN, Tkachuk VA, Vyssokikh MY, Yaguzhinsky LS, Zorov DB. An attempt to prevent senescence: a mitochondrial approach. Biochim Biophys Acta. 2009 May;1787(5):437-61. Epub 2008 Dec 29. Review. PubMed PMID: 19159610.

5: Obukhova LA, Skulachev VP, Kolosova NG. Mitochondria-targeted antioxidant SkQ1 inhibits age-dependent involution of the thymus in normal and senescence-prone rats. Aging (Albany NY). 2009 Apr 22;1(4):389-401. PubMed PMID: 20195490; PubMed Central PMCID: PMC2830050.

6: Blüher M, Kahn BB, Kahn CR. Extended longevity in mice lacking the insulin receptor in adipose tissue. Science. 2003 Jan 24;299(5606):572-4. PubMed PMID: 12543978.

7: Odens M. Prolongation of the life span in rats. J Am Geriatr Soc. 1973 Oct;21(10):450-1. PubMed PMID: 4729008.

8: Lemon JA, Boreham DR, Rollo CD. A complex dietary supplement extends longevity of mice. J Gerontol A Biol Sci Med Sci. 2005 Mar;60(3):275-9. PubMed PMID: 15860460.

9: Kitani K, Minami C, Isobe K, Maehara K, Kanai S, Ivy GO, Carrillo MC. Why (--)deprenyl prolongs survivals of experimental animals: increase of anti-oxidant enzymes in brain and other body tissues as well as mobilization of various humoral factors may lead to systemic anti-aging effects. Mech Ageing Dev. 2002 Apr 30;123(8):1087-100. Review. PMID: 12044958 [PubMed - indexed for MEDLINE]

10: Kitani K, Kanai S, Ivy GO, Carrillo MC. Assessing the effects of deprenyl on longevity and antioxidant defenses in different animal models. Ann N Y Acad Sci. 1998 Nov 20;854:291-306. Review. PMID: 9928438 [PubMed - indexed for MEDLINE]

11: Kokkonen GC, Barrows CH: The effect of dietary vitamin, protein and intake levels on the life span of mice of different ages. AGE.1985 Jan;8(1): 13-17.

12: Masoro EJ. A forum for commentaries on recent publications. FIRKO mouse report: important new model--but questionable interpretation. J Gerontol A Biol Sci Med Sci. 2003 Oct;58(10):B871-2. PubMed PMID: 14570851.

13: Anon. The stuff on which quackery thrives? Nutr Rev. 1974 Oct;32(10):316-7. PubMed PMID: 4416514.

14: Dhahbi JM, Kim HJ, Mote PL, Beaver RJ, Spindler SR. Temporal linkage between the phenotypic and genomic responses to caloric restriction. Proc Natl Acad Sci U S A. 2004 Apr 13;101(15):5524-9. Epub 2004 Mar 25. PubMed PMID: 15044709; PubMed Central PMCID: PMC397416.

15: Spindler SR, Mote PL. Screening candidate longevity therapeutics using gene-expression arrays. Gerontology. 2007;53(5):306-21. Epub 2007 Jun 15. Review. PubMed PMID: 17570924.

16: Spindler SR. Review of the literature and suggestions for the design of rodent survival studies for the identification of compounds that increase health and life span. Age (Dordr). 2011 Mar 22. [Epub ahead of print]. PMID: 21424790