Frailty Assessment in an Aging Mouse Model

Summary

The goal of the present protocol is to provide instructions and guidelines for assessing physical frailty in C57Bl/6x129J laboratory mice. Specific details regarding each measure used in the physical frailty index are explained. Several methods for reporting frailty results are also presented.

Abstract

A frailty index (FI) is a powerful method of assessing animal models of aging, serving as a proxy measure of biologic age and health span. Frailty represents a state of increased vulnerability to adverse outcomes due to the accumulation of health deficits that limit independence. The concept is valuable for animal studies that inform human geroscience. For example, using FI scores, animals with a similar chronological age can be differentiated by their biological age. Thus, FI can be used in preclinical studies to measure biological age, track frailty longitudinally, and assess health span.

A murine frailty index was recently developed using 31 health-related deficits. However, its length and requirement for specialized equipment pose limitations in practical use. Here, we present an adapted 16 item frailty index that excludes parameters requiring special instrumentation or invasive testing. The selected deficits are easily observable or measurable and evaluate integument, musculoskeletal, neuromuscular, sensory, urogenital, and respiratory systems. Body weight is measured for longitudinal tracking but excluded from the FI calculation. Each deficit is scored as 0, 0.5, or 1, based on severity, with the FI computed as the mean of the 16 deficit scores.

This streamlined FI allows rapid assessment of large cohorts, can be applied longitudinally to the same animals, and requires no specialized equipment. We demonstrate its feasibility in C57BL/6×129 mice, showing age-associated increases in frailty, variation among individual mice, and the ability to detect deficits across multiple systems. This tool enables laboratories to incorporate frailty into diverse experimental designs, provides a practical approach for assessing health span, and enhances translational relevance in geroscience. This frailty index is adapted from previously validated mouse clinical frailty methodologies and is intended for straightforward, longitudinal use alongside ongoing studies.

Introduction

Frailty comprises increased susceptibility to adverse health outcomes typically associated with advanced age¹,^2,3. It presents as decreased resilience to stressors and loss of normal physiological function, potentially limiting independence and predisposing older individuals to co-morbidities and mortality^3,4,5. Frailty may help explain the differences in health outcomes commonly seen between those of the same chronological age⁶. In humans, frailty assessments include the frailty phenotype^7,8and the frailty index^9,10,11, which assess physical performance (e.g., weakness, endurance, or weight loss) and the number of deficits across organ systems, respectively. Animal studies have incorporated a five-physical-criterion frailty score as well as a 31 item frailty index^12,13,14. Both approaches have their strengths and weaknesses. Frailty phenotyping allows excellent characterization of decreased physical performance, of value for projects interested in sarcopenia, neuromotor dysfunction, or any other aging-related change in physical function. Phenotyping is non-invasive and non-terminal. However, this method of quantifying frailty requires specialized equipment (e.g., rodent treadmills, rotarods, running wheels, and grip meters) and is very time-consuming. A frailty phenotype focusing on physical function lacks the robustness of indices that consider frailty across various body systems. Accordingly, frailty indices developed to assess deficits across multiple systems create a more comprehensive rating of frailty. They are also generally non-invasive, easy to administer, and do not require specialized equipment or extensive time. Frailty indices based on deficit accumulation do not readily assess cognitive function and introduce subjectivity in scoring deficits, particularly if multiple evaluators are used, though the effects of this subjectivity can be minimized through discussing and refining assessment techniques¹⁵.

The goal of the present method is to quantify frailty using a simple, practical tool, allowing for frailty to be added as a longitudinal outcome to existing and future projects. Multiple considerations were made when developing a frailty measure to fit this goal, one being the reliance on specialized equipment. Several frailty phenotyping methods have been proposed but they require equipment that may be inaccessible to some or impractical for frequent use. For example, open-field monitoring is used in some frailty phenotyping studies to measure exploratory behaviors and voluntary physical activity^16,17. Phenotyping by open-field monitoring requires both specialized camera and computer equipment and considerable time commitment as the mice individually undergo the acclimation and observation periods. Other phenotypes require the use of specialized equipment like rodent treadmills and Rotarods^13,18,19. The demands that a suite of tests, including these and other measures, places on animal subjects are too great for the frequent, repeated tests needed in a longitudinal study design, not to mention the animal acclimation and investigator training required for each. So, while not aligned with our goal, frailty phenotyping and the common tests used can be applicable to experiments requiring cross-sectional and infrequent longitudinal observation.

Frailty indices are generally non-invasive, are practical to integrate into projects, and can be time efficient^12,20. Using an index also allows for details to be drawn from existing methodologies that have been shown to be reproducible and reliable^15,21. In collaboration with multiple veterinary staff with experience assessing mouse frailty, an abbreviated list of criteria/deficits was selected from a larger list¹², including criteria of the integument (alopecia, loss of fur color, dermatitis, coat condition), musculoskeletal (kyphosis, tail stiffening, tumors, distended abdomen, body condition score), neuromuscular (gait disorders, tremor), sensory (cataracts, eye discharge/swelling), urogenital (rectal and vaginal/penile prolapse), and respiratory (breathing rate/depth) systems. These deficits were chosen specifically as they cover a comprehensive range of systems and can be evaluated without equipment or specialized examinations beyond open-field and manual examination. The specific list these are drawn from has been clinically validated¹² and confirmed to have high inter-rater reliability as found by multiple laboratory groups^15,21. This was an important consideration given the subjective nature of many of the chosen deficits. Reasons for deficits to be excluded from the abbreviated index included requirement of some form of equipment (grip strength, temperature) specialization beyond the desired scope of this simple index (loss of whiskers, vestibular disturbance, corneal opacity, vision and hearing loss, microphthalmia, nasal discharge, piloerection), and avoidance of institutional humane endpoints (diarrhea, malocclusions, mouse grimace scale). The present tool, therefore, represents a pragmatic subset of the validated 31 item index, tailored to minimize equipment needs and time burden while maintaining multi-system coverage. It is important to note that the present protocol describes one such way a larger frailty index can be adapted to fit the goals of a given experiment, serving as a guide for investigators interested in simplified or customized tools for assessing mouse frailty. Body weight was also included in the assessment, but not in the estimation of the frailty index, as conflicting reports suggest variable impact of both weight gain and loss on frailty^12,14,22,23. The purpose of shortening the list of deficits used in determining the frailty index was to highlight the flexibility and adaptability of such an index while simplifying the measurement tool to the selected criteria and retaining the comprehensive assessment of frailty in C57Bl/6x129J mice. This change allows for a complete assessment of frailty without equipment-intensive methods or complicated examination. With some training, investigators can expect this comprehensive assessment of physical frailty to take less than 2 min for a single mouse compared to nearly 4 min for a longer, 31 item index¹².

Protocol

All mice (C57Bl/6x129J) were bred at colonies maintained at the Mayo Clinic. Mice were group housed with littermates by sex, maintained on a 12 h light-dark schedule under specific pathogen-free conditions with ad libitum access to food and water. A convenience sample of mice (n = 46; 36 females) aged 12 to 30 months was evaluated for frailty every 2 weeks for eight months. All protocols and animal care guidelines were approved by the Institutional Animal Care and Use Committee at the Mayo Clinic (protocol A00003349, approved 5 October 2023), in compliance with National Institute of Health Guidelines. Criteria for euthanasia for humane endpoints included weight loss greater than or equal to 20% of body weight, inability to ambulate, inability to reach food and/or water, and tumors including single tumors measuring 2 cm at the longest point of the tumor, multiple tumors with cumulative tumor size (the sum of the longest point of each tumor) equaling 2 cm, or tumor(s) that ulcerate or lose the integrity of the skin covering the tumor for any reason.

1. Setup

NOTE: During the physical frailty assessment, ensure a clean and hazard-free workspace for handling mice and wear proper personal protection equipment.

1. Collect the necessary equipment-Scale, notebook or laptop for recording data, and a mouse cage.

2. Physical frailty assessment

1. Divide the assessment into three parts-open field examination, manual examination, and weighing. Complete assessments at a consistent location and time of day to minimize anxiety- or ambient-related effect on deficits.
   1. Open field examination: Transfer the mouse of interest into an open environment and examine several physical characteristics, including the presence of alopecia, loss of fur color, dermatitis, poor coat condition, kyphosis, tail stiffening, gait disorders, tremor, and abnormal breathing.
   2. Manual examination: Gently scruff the mouse to allow the experimenter to further examine physical characteristics, including the presence of alopecia, loss of fur color, dermatitis, tumors, distended abdomen, cataracts, eye discharge, rectal prolapse, vaginal/uterine/penile prolapse, and poor body condition score.
      NOTE: Because scruffing is inherently stressful, alternative handling methods such as cupping or tube restraint can be used during transfers and some assessments to minimize animal discomfort and better align with institutional policies.
   3. Following open field and manual examinations, weigh the mouse. Zero the scale between each animal and record weights to the nearest 0.1 g. Again, ensure time-of-day consistency of the assessment to minimize natural circadian inconsistencies.
2. Following the assessment of each mouse cage, clean the scale and the open field by spraying surfaces with disinfectant or other appropriate cleaning agent and wiping with a paper towel.

3. Examination, including the following 17 characteristics and behaviors

1. Alopecia-signs of hair loss and/or thinning
   1. Assess alopecia during both open field and manual examination. Common areas of hair loss and thinning include the back of the neck/upper back, neck and armpits, and stomach. Note that there are natural areas of thinner hair on the underside of mice, especially near joints. Count alopecia only if hair loss and thinning are beyond that of natural patterns observed in young, non-frail mice.
   2. Score alopecia as follows: 0 = normal fur density; 0.5 ≤ 25% of body surface displaying fur loss; 1.0 ≥ 25% of body surface displaying fur loss
2. Loss of fur color-change in fur color, e.g., from black to grey, brown, or white
   1. Assess loss of fur color during both open field and manual examination. Changes in fur color often begin on the underside of the mouse but will progress to whole body changes with progressing frailty.
   2. Score loss of fur color as follows: 0 = normal color; 0.5 = patchy, focal grey, brown, or white changes; 1.0 = grey, brown, or white fur on >25% of body surface
3. Dermatitis-presence of skin lesions
   1. Assess dermatitis during both open field and manual examination. Presence of any skin lesions indicating dermatitis, with most focal lesions found on the neck, flanks, and under the chin.
   2. Score dermatitis as follows: 0 = absent; 0.5 = focal lesions of small size; 1.0 = multifocal or widespread lesions
4. Coat condition-signs of poor grooming
   1. Assess coat condition during open field examination. Normal coat condition, usually present in young, non-frail mice, includes a smooth, sleek, shiny coat. Ruffled, matted, and/or ungroomed fur are signs of poor coat condition and often accompany alopecia and changes in fur color.
   2. Score coat condition as follows: 0 = smooth, sleek, shiny coat; 0.5 = slightly ruffled; 1.0 = unkempt and ungroomed, matted appearance
5. Kyphosis-presence of curvature of the spine or hunched posture
   1. Assess kyphosis during the open field examination. Kyphosis is most often present in the upper back and will present as a clear concave curvature beyond that seen in any young, non-frail mice. To confirm the presence of kyphosis, palpate the upper spine to examine for a kyphotic bend. Look for a permanently hunched posture following kyphosis that will be noticeable as a whole-body hunch as opposed to a more local kyphotic bend.
   2. Score kyphosis as follows: 0 = no cervical to thoracic spinal curvature (approximately 180° to 150°); 0.5 = slight cervical to thoracic spinal curvature (approximately 150° to 130°); 1.0 = significant curvature (<130°) and associated hunched posture
6. Tail stiffening-lack of curling response to light touch
   1. Assess tail stiffening during open field examination. While the mouse is freely moving, gently stroke the tail with a finger. The tail of a healthy mouse will wrap around the finger when it is touched. This is best observed when the mouse is relaxed.
   2. Score tail stiffening as follows: 0 = none; 0.5 = tail responsive but does not curl; 1.0 = tail unresponsive to touch
7. Gait disorders-abnormalities during locomotion
   1. Assess the presence of gait disorders during open-field examination. Common changes to the gait include dragging limbs, circling, wobbling, and a wide stance. Use climbing ability to assess gait by placing the mouse on a large cage lid, tilting the lid, and lightly brushing the rear of the mouse to motivate climbing.
   2. Score gait disorders as follows: 0 = none; 0.5 = abnormal gait but mouse can walk; 1.0 = marked gait abnormality that impairs ability to move freely
8. Tremor-unprovoked trembling or shaking
   1. Assess tremor during open-field observation. Monitor the mouse during rest and movement for the presence of a tremor, which usually presents as paw shaking.
   2. Score tremor as follows: 0 = no tremor; 0.5 = paw shake or tremor during ambulation or high levels of activity; 1.0 = marked tremor with impaired ability to move freely
9. Breathing rate/depth-abnormal respiratory effort
   1. Assess breathing rate/depth during open-field examination. Any changes from normal, regular breathing at rest, as observed in a young, non-frail mouse, are scored. Usually, changes include an increased rate and decreased depth of breathing.
   2. Score breathing rate/depth as follows: 0 = normal; 0.5 = modest change in rate/depth; 1.0 = marked change in rate/depth, or gasping.
10. Tumors-presence of abnormal masses
    1. Assess the presence of tumors during manual examination. While scruffing the mouse, examine the mouse for visible tumors and run a finger along the underside of the mouse to check for palpable tumors. Visible asymmetry is a sign of potential tumors. The presence of tumors can be confirmed post mortem if possible.
    2. Score tumors as follows: 0 = none; 0.5 = one tumor of less than 1 cm³ in volume; 1.0 = single tumor greater than 1 cm³ in volume or multiple smaller tumors
11. Distended abdomen-presence of excess bulge below the rib cage
    1. Assess the distended abdomen during manual examination. While scruffing the mouse, assess the presence of excess bulging or abdominal fluid. Look for the presence of a slight bulge below the rib cage to a pronounced W-shaped bulge in the abdomen and palpable during examination.
    2. Score distended abdomen as follows: 0 = none; 0.5 = slight bulge; 1.0 = clear distension, W-shaped bulge below ribs
12. Cataracts-presence of cloudy eyes/white spots
    1. Assess cataracts during manual examination. While scruffed, evaluate the mouse's eyes for clouding of the cornea. Complete clouding of at least one eye is counted as 1.0.
    2. Score cataracts as follows: 0 = none; 0.5 = small opaque spot/changes to one or both corneas; 1.0 = significant clouding/spotting of one of both corneas
13. Eye discharge-buildup of bodily fluid around the eyes
    1. Assess eye discharge during manual examination. While scruffed, evaluate the mouse's eyes for evidence of secretions, crusting or discoloration around the edges of the eyes. Discharge is usually dried, associated with swelling, and will result in changes to the normal, sleek, black eye color seen on young, non-frail mice.
    2. Score eye discharge as follows: 0 = normal; 0.5 = slight swelling of and/or secretions around one or other eyes; 1.0 = significant swelling of and/or secretions around one or both eyes
14. Rectal prolapse-abnormal extrusion of the rectum
    1. Assess rectal prolapse during manual examination. While scruffed, examine for signs of prolapsed tissue in the absence of evident straining. Score any rectal tissue prolapsed out of the anus.
    2. Score rectal prolapse as follows: 0 = no prolapse; 0.5 = some prolapsed tissue, healthy looking; 1.0 = significant prolapsed tissue, unhealthy tissue.
15. Vaginal/uterine/penile prolapse-abnormal extrusion of genitalia
    1. Assess vaginal/uterine/penile prolapse during manual examination. While scruffed, examine for signs of prolapsed tissue in the absence of evident straining. Score any genitalia prolapse.
    2. Score vaginal/uterine/penile prolapse as follows: 0 = no prolapse; 0.5 some prolapsed tissue, healthy looking; 1.0 = significant prolapsed tissue, unhealthy tissue.
       NOTE: Prolapse can occur due to distress from handling, so if a deficit is suspected, place the mouse back onto the open field, lifting the tail to confirm the presence of prolapsed tissue.
16. Body condition score-presence of excess or minimal fat and evidence of loss of muscle mass
    1. Assess body condition score during manual examination. While scruffing the mouse, take note of the amount of fat covering the lower back and pubic bone; frailty will usually present a loss of fat in this region. Moderate loss of fat resulting in more prominent bones than young, non-frail mice will be scored as 0.5. Once the bones become very prominent, even sharp, or there is evident loss of muscle mass, the score will be 1.0.
    2. Score body condition score as follows: 0 = bones palpable but not prominent; 0.5 = bones prominent and/or barely felt; 1.0 = bones very prominent and/or not felt.
17. Weight-assessment at a consistent time of day (e.g., within 2 to 4 h of the beginning of the inactive period) following the open-field and manual examination.
    1. Assess weight following frailty measures.

4. Implementing the physical frailty assessment

NOTE: Much of the implementation of this assessment should be customized to the needs of the experimenter and their experimental goals. Details provided here can serve as an example of how one might implement the frailty assessment and provide some helpful guidance for general purposes.

1. Frequency: Once a cohort or colony of mice to be assessed is identified, make physical frailty assessments every 2 weeks. Keep the time and day of the assessments consistent during the evaluation period.
2. Data Recording: During the evaluation of each mouse, assess a score (0, 0.5, or 1.0) to each of the frailty measures and record a weight; use an average of the scores of all sixteen deficits is used as a final frailty score. Take any relevant notes regarding the health and behavior of the mice, including changes to any of the measures that will require increased attention during subsequent evaluations and abnormal posthandling behaviors. Perform no rescoring for prior scores. Compute the frailty index as the arithmetic mean of the 16 deficit scores (0/0.5/1.0), expressly excluding body weight from the calculation.
   NOTE: It is vital to ensure the correct data is being ascribed to the correct mouse over the course of the longitudinal evaluation period.

Results

A convenience sample of naturally aging C57Bl/6x129J mice was assessed for frailty using the index described here. Each mouse (n = 46; 36 females) over 12 months of age was assessed every 2 weeks, with the evaluation period lasting approximately 6 months. A cross-sectional cohort at four ages of interest (12, 18, 24, and 30 months) was selected to assess frailty index scoring across age groups representing 100%, 95%, 80% and 30% survival, respectively²⁴. T...

Discussion

Critical to the success of this frailty index is consistency when evaluating the many subjective criteria that may be present with deficits. Several steps can be taken to ensure the quality of results and overall reliability of this index. The same investigator can be used during observation, ensuring that the same judgement of deficit score will be given across animals and time points. If experiment design or logistics prevent this consistency, each investigator should independently familiarize themselves with the index...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Digital Scale (multi-unit)	Tree	KHR3001
Peroxigard Ready to Use	Virox Technologies Inc	PRTU242101	disinfectant