ABT263 treatment in naturally aged mice

Validation Score: 0.950 Price: $0.50 natural aging naturally aged mice Status: proposed

What This Experiment Tests

Validation experiment designed to validate causal mechanisms targeting N/A in naturally aged mice. Primary outcome: rejuvenation of aged HSCs and MuSCs through senescent cell depletion

Description

Naturally aged mice were treated with ABT263 to evaluate its senolytic effects in normal aging conditions. The study assessed whether ABT263 could effectively deplete accumulated senescent cells in aged mice, including senescent bone marrow hematopoietic stem cells and muscle stem cells. The research examined the rejuvenation potential of aged tissue stem cells following senescent cell clearance and evaluated the overall anti-aging effects of the treatment.

TARGET GENE

N/A

MODEL SYSTEM

naturally aged mice

ESTIMATED COST

TIMELINE

0 months

PATHWAY

BCL-2/BCL-xL apoptosis pathway, stem cell aging pathways

SOURCE

extracted_from_pmid_26657143

PRIMARY OUTCOME

rejuvenation of aged HSCs and MuSCs through senescent cell depletion

Scoring Dimensions

📖 Wiki Pages

Apoptosis Pathway in Neurodegenerationmechanism

Protocol

Study Design

Animal Model: C57BL/6J mice, naturally aged (18-22 months), both sexes
Drug: ABT263 (S6381, Selleck Chemicals), 50 mg/kg dissolved in 10% DMSO, 40% PEG300, 5% Tween-80, 45% sterile water
Control: Vehicle-only formulation
Group Size: n=12 per group (vehicle, ABT263), based on power analysis (α=0.05, β=0.2, expected effect size d=1.2)

Phase 1: Baseline Assessment (Day -7)

Timepoint: 7 days before treatment initiation

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Study Design

Animal Model: C57BL/6J mice, naturally aged (18-22 months), both sexes
Drug: ABT263 (S6381, Selleck Chemicals), 50 mg/kg dissolved in 10% DMSO, 40% PEG300, 5% Tween-80, 45% sterile water
Control: Vehicle-only formulation
Group Size: n=12 per group (vehicle, ABT263), based on power analysis (α=0.05, β=0.2, expected effect size d=1.2)

Phase 1: Baseline Assessment (Day -7)

Timepoint: 7 days before treatment initiation

Age-matched young controls (3-4 months, n=8) and aged baseline cohort (n=24) undergo baseline phenotyping
HSC harvest: Flush femurs and tibias with PBS + 2% FBS; enrich for lineage-negative cells (Miltenyi Biotec Lineage Cell Depletion Kit)
MuSC isolation: Digest gastrocnemius muscles with collagenase II (800 U/mL) + dispase II (2.4 U/mL); enrich via Pax7-GFP reporter or anti-α7-integrin magnetic sorting
Flow cytometry panels:
HSC: LSK (Lin−Sca-1+c-Kit+), CD48/CD150, CD34/CD16/32 (myeloid bias)
MuSC: α7-integrin+CD34+, Pax7, Ki-67
Senescence markers: SA-β-gal activity (C12FDG staining), p16INK4a reporter (Luciferase or qPCR: Cdkn2a)
SASP multiplex: IL-6, TNF-α, CXCL1, MMP-3 (Luminex or ELISA)
Record body weight, grip strength, rotarod performance

Phase 2: ABT263 Treatment (Days 0, 2, 4)

Dosing regimen: Oral gavage, 50 mg/kg, every other day × 3 doses

Randomize aged mice into vehicle (n=12) and ABT263 (n=12) groups
Administer via oral gavage (20G curved catheter) in morning
Monitor daily for adverse effects (weight loss >15%, hunched posture, decreased mobility)
Collect plasma at 2h and 6h post-dose on Day 0 for pharmacokinetics (LC-MS/MS)

Phase 3: Acute Senescent Cell Clearance (Days 5-7)

Endpoint: 24-72 hours after final dose

Senescent cell burden (n=4 per group sacrificed):

SA-β-gal tissue staining: Liver, lung, adipose, muscle, bone marrow
p16INK4a immunofluorescence: Tissue sections co-stained with DAPI
TUNEL assay: Quantify apoptotic cells in senescent cell populations
SASP plasma levels: Compare pre- vs post-treatment

Off-target toxicity assessment:

Complete blood count (CBC) via IDEXX ProCyte Dx
Liver function: ALT, AST, bilirubin (Beckman Coulter AU680)
Kidney function: BUN, creatinine
Histopathology: H&E staining of liver, kidney, lung, heart

Phase 4: Early Regeneration Phase (Days 14-21)

Stem cell function assays (n=8 per group):

HSC function:

Competitive repopulation assay: Transplant 1×10^6 CD45.2 donor bone marrow cells (aged vehicle or ABT263) + 1×10^6 CD45.1 competitor cells into lethally irradiated (900 cGy) CD45.1 recipients
Analyze peripheral blood chimerism at 4, 8, 12, 16 weeks post-transplant (flow cytometry: CD45.2 vs CD45.1)
Lineage distribution: B220 (B cells), CD3 (T cells), Gr-1/Mac-1 (myeloid)
Serial transplantation: Collect bone marrow from primary recipients at 16 weeks; transplant into secondary recipients

MuSC function:

Notexin-induced muscle regeneration: Inject 10 μL of 10 μM notexin into tibialis anterior; harvest at 7 and 14 days post-injury
Muscle histology: H&E staining to assess central nucleation (regenerating fibers), fiber cross-sectional area (CSA), fibrosis (Masson's trichrome)
MuSC quantification: Pax7+ cell density per 100 fibers
Functional recovery: Grip strength and running wheel activity at 7, 14, 21 days post-injury

Phase 5: Long-Term Rejuvenation (Weeks 8-12)

Final endpoints (remaining n=8 per group, plus young controls):

Hematopoietic parameters:

Complete blood count with differential
Bone marrow cellularity (femur + tibia total nucleated cells)
HSC frequency and absolute numbers: LSK, LT-HSC (CD150+CD48−), ST-HSC (CD150−CD48−)
Myeloid:lymphoid ratio in bone marrow

MuSC parameters:

MuSC yield per gram muscle (gastrocnemius, quadriceps)
Cell cycle analysis: Ki-67/DAPI flow cytometry
Mitochondrial function: Seahorse XF analyzer (OCR/ECAR in MuSC culture)
Telomere length: qFISH or Flow-FISH

Systemic markers:

Inflammatory cytokines: IL-6, TNF-α, IL-1β, IFN-γ (Luminex)
Metabolic panels: fasting glucose, insulin (ELISA), cholesterol
Physical function: rotarod latency, hanging wire duration, stride length analysis

Phase 6: Mechanistic Studies (Parallel, Weeks 4-8)

BH3 profiling (Bio-techne or in-house):

Measure apoptotic threshold in aged vs young HSCs and MuSCs
Determine Mcl-1, Bcl-2, Bcl-xL expression (Western blot: Bcl-2, Bcl-xL, Mcl-1, NOXA, BIM)
Cyto-ID flow cytometry for autophagic flux

Senescent cell engraftment model (validation):

Transplant DiR-labeled senescent fibroblasts (γ-irradiated, p16-high) into aged mice ± ABT263
Track clearance via IVIS imaging at 24, 48, 72h post-transplant

Expected Outcomes

Senescent cell clearance: ABT263 will reduce SA-β-gal+ cells by 40-60% in liver, lung, and adipose tissue, and reduce p16INK4a+ cells in bone marrow by 35-50% compared to vehicle-treated aged mice.

HSC functional rejuvenation: In competitive repopulation assays, aged ABT263-treated donors will show 2.5-3.5-fold higher engraftment efficiency at 16 weeks compared to aged vehicle donors, reaching 60-75% of young donor engraftment levels.

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Success Criteria

Senescent cell clearance: ABT263 reduces SA-β-gal+ cells by ≥35% in at least 3 of 5 tissues (liver, lung, adipose, muscle, bone marrow) compared to vehicle controls (two-tailed t-test, p<0.01).
HSC engraftment: Donor cell chimerism in peripheral blood at 16 weeks post-transplant reaches ≥50% for ABT263-treated aged donors, significantly different from vehicle (≤25%) with p<0.001 (Mann-Whitney U test) and effect size r≥0.7.

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