Chronic inflammation induces telomere dysfunction and accelerates ageing in mice methods
Aim. Evidence-backed execution summary for Chronic inflammation induces telomere dysfunction and accelerates ageing in mice methods from Chronic inflammation induces telomere dysfunction and accelerates ageing in mice.
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mouse
Subject model for the experiment.
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Chronic inflammation reinforces cellular senescence
reagent used in the protocol.
- Use
- To understand the mechanisms by which chronic pro-inflammatory signals increased senescence-associated mitochondrial ROS production we first tested the role of p38MAPK. Signalling through p38MAPK drives enhanced mitochondrial ROS and activates NF-κB in senescence. Accordingly, inhibition of p38MAPK using SB2035...
Impaired tissue regeneration in nfkb1 -/- mice
reagent used in the protocol.
- Use
- NF-κB-mediated inflammation can stimulate cell proliferation, especially of tumour cells. Conversely, NF-κB is also involved in apoptosis signalling, and NF-κB-driven signalling can stabilize senescent cell arrest in vitro. To understand how NF-kB-driven chronic inflammation can cause accelerated ag...
Impaired tissue regeneration in nfkb1 -/- mice
reagent used in the protocol.
- Use
- The gut epithelium is constantly regenerated from stem cells located at the bottom of the crypts. Low mucosal thickness of the colon and low villus length in the intestine are early morphological indicators of decreasing stem/progenitor function in the ageing gut, leading to malabsorption. Both parameters were decr...
Chronic inflammation reinforces cellular senescence
reagent used in the protocol.
- Use
- Senescence is not a cell-autonomous process. By secreting bioactive molecules including interleukins, chemokines and ROS, senescent cells induce powerful bystander effects that spread senescence to neighbouring normal cells. Given that both ROS and SASP signals are stronger in nfkb1 -/- cells, we hypot...
Feedback between inflammation and telomere dysfunction in vivo
reagent used in the protocol.
- Use
- Having shown enhanced telomere dysfunction and senescence in two different models of chronic inflammation in vivo, we investigated whether conversely telomere dysfunction would lead to a pro-inflammatory state, for example, in tissues from late-generation terc -/- mice. This was in fact the case: NF-...
Methods
reagent used in the protocol.
- Use
- Experiments were performed on male nfkb1 -/- mice on a pure C57Bl/6 background and C57Bl/6 wt controls. Pure background mice were a gift from Jorge Caamano, (Birmingham University, UK). Experiments were performed at 36 weeks of age if not indicated otherwise. Immunohistochemistry was also performed on mi...
Cell culture
reagent used in the protocol.
- Use
- Ear clippings were transported and stored (not longer than 1 h) in DMEM containing serum on ice. Punches were washed three times with serum-free media, finely cut and incubated for 2-3 h at 37 °C in 2 mg ml -1 collagenase A in DMEM. A single-cell suspension was obtained...
Cell culture
reagent used in the protocol.
- Use
- Cells were treated with 10 nM siRNA (Qiagen, SI101392391, SI01392398 or no. 1027281) at 1 day before IR in HiPerFect (Qiagen). Ibuprofen (Sigma, no. I7905-1G, 0.2 mM) was added to the cell culture media immediately after IR. Inhibitors were used from immediately after IR at the following concentrations:...
Feedback between inflammation and telomere dysfunction in vivo
Together these data suggest that there exists a positive feedback loop system between telomere dysfunction, senescence-associated ROS production and pro-inflammatory signalling that induces and stabilizes senescence in vivo, which in turn limits regenerative capacity of tissues.
- Use
- Together these data suggest that there exists a positive feedback loop system between telomere dysfunction, senescence-associated ROS production and pro-inflammatory signalling that induces and stabilizes senescence in vivo, which in turn limits regenerative capacity of tissues.
Ibuprofen and BHA treatment
For liver and gut regeneration studies, mice were given ibuprofen mixed in their food to a daily dosage of 50 mg per kg (mouse) per day. Treatment started at 8 weeks of age for 4 consecutive weeks. For senescence studies, mice received ibuprofen via pump (mini-osmotic pump, Alzet, model 2004) for a period of 8...
- Use
- For liver and gut regeneration studies, mice were given ibuprofen mixed in their food to a daily dosage of 50 mg per kg (mouse) per day. Treatment started at 8 weeks of age for 4 consecutive weeks. For senescence studies, mice received ibuprofen via pump (mini-osmotic pump, Alzet, model 2004) for a period of 8...
Cell culture
Cells were treated with 10 nM siRNA (Qiagen, SI101392391, SI01392398 or no. 1027281) at 1 day before IR in HiPerFect (Qiagen). Ibuprofen (Sigma, no. I7905-1G, 0.2 mM) was added to the cell culture media immediately after IR. Inhibitors were used from immediately after IR at the following concentrations:...
- Use
- Cells were treated with 10 nM siRNA (Qiagen, SI101392391, SI01392398 or no. 1027281) at 1 day before IR in HiPerFect (Qiagen). Ibuprofen (Sigma, no. I7905-1G, 0.2 mM) was added to the cell culture media immediately after IR. Inhibitors were used from immediately after IR at the following concentrations:...
Crosslinked chromatin immunoprecipitation assay
ChIP assay was carried out using crosslinked chromatin prepared from wt, terc -/- and nfkb1-/- livers. Briefly, the chromatin was prepared by resuspending powdered, frozen liver in 10 ml cold PBS with protease inhibitors and crosslinked with 1% formaldehyde for 5 min. The reaction...
- Use
- ChIP assay was carried out using crosslinked chromatin prepared from wt, terc -/- and nfkb1-/- livers. Briefly, the chromatin was prepared by resuspending powdered, frozen liver in 10 ml cold PBS with protease inhibitors and crosslinked with 1% formaldehyde for 5 min. The reaction...
mRNA expression analysis
RNA was isolated from frozen tissue using RNeasy Mini Kit and QIAshredder. RNA quality was checked on the Bioanalyzer. Total RNA was reverse-transcribed using Omniscript Reverse Transcription Kit (Qiagen). QPCR was run in triplicates using the following primers:
- Use
- RNA was isolated from frozen tissue using RNeasy Mini Kit and QIAshredder. RNA quality was checked on the Bioanalyzer. Total RNA was reverse-transcribed using Omniscript Reverse Transcription Kit (Qiagen). QPCR was run in triplicates using the following primers:
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Accelerated ageing in nfkb1 -/- mice
As chronic inflammation had been linked to ageing, we investigated whether nfkb1 -/- mice might age faster than their wt counterparts. We tested a wide range of biomarkers of mouse ageing in nfkb1 -/- mice at 36 weeks of age, their C57Bl/6 littermates at the same age and in old C57Bl/6 mice at 104 and 132 weeks ( ). With the exception of indicators of muscle weakness (ataxia, sarcopenia and kyphosis) and of spontaneous tumours, all tested biomarkers of ageing were more frequently positive in 36-week-old nfkb1 -/- mice as in their wt counterparts and often reached levels similar to wt mice aged ≥2 years ( ). Both mean and maximum lifespan were reduced in nfkb1 -/- mice ( ). At ~\n36 weeks of age, adult nfkb1 -/- mice showed frequent hair loss, early hair greying (starting at ~\n20 weeks of age), scruffy fur and cachexia...
Impaired tissue regeneration in nfkb1 -/- mice
The gut epithelium is constantly regenerated from stem cells located at the bottom of the crypts. Low mucosal thickness of the colon and low villus length in the intestine are early morphological indicators of decreasing stem/progenitor function in the ageing gut, leading to malabsorption. Both parameters were decreased in nfkb1 -/- mice aged 36 weeks ( ) to values similar to those in 52-104-week-old wt mice. This was not associated with enhanced apoptosis ( ), suggesting a decreased regenerative capacity in either stem or progenitor cells. To address this, we isolated intestinal crypts from 12- and 54-week-old mice and analysed their growth in organotypic culture over a 10-day observation period ( ). We measured both the frequencies of crypts that started to grow, which is indicative of stem cell function, and growth rates, indicative of proliferation primarily in...
Chronic inflammation reinforces cellular senescence
Mouse fibroblasts senesce spontaneously after few population doublings in 21% ambient oxygen as a consequence of a stress-induced DDR. Mouse adult ear fibroblasts (MAFs) from nfkb1 -/- donors senesced faster than their wt counterparts as indicated by accelerated loss of proliferative capacity ( ) and increased expression of the senescence marker senescence-associated β-galactosidase (sen-β-gal; ). To address the underlying mechanisms of accelerated senescence in nfkb1 -/- cells, we used a well-established model of induction of cell senescence by ionizing radiation (IR). Radiation-induced DDR can activate NF-κB through an ATM-dependent mechanism. However, the immediate response to DNA damage was not different between nfkb1 -/- and wt MAFs as shown by equal activation of ATM ( and ) and p53 ( ) and equal frequencies of DNA damage foc...
Chronic inflammation reinforces cellular senescence
It is well established that the induction of the senescent phenotype including sen-β-Gal, SASP and enhanced ROS production requires at least 3-7 days following IR. Multiple feedback loops interconnecting the DDR with SASP and ROS phenotypes via p38MAPK and NF-κB have been described, all of them stabilizing senescence with some kinetic delay. Accordingly, after a sufficient delay following IR, a wide range of senescence markers was elevated in both wt and nfkb1 -/- MAFs. Importantly, the senescent phenotype was aggravated in nfkb1 -/- cells according to every single marker tested ( and ). Specifically, there were more sen-β-Gal-positive cells ( ), more DNA damage foci per nucleus ( ), more mitochondrial superoxide was produced per cell ( ), more ROS accumulated in the cytoplasm ( ) and the expression of the CDKN1A and CDKN2A genes encoding...
Feedback between inflammation and telomere dysfunction in vivo
Telomere dysfunction is an important driver of cell senescence, and TAF ( ) are established as markers of telomere dysfunction and cell senescence in human and mouse tissues. TAF frequencies increased with age in liver hepatocytes and in enterocytes in intestinal crypts from wt mice ( ) similar to other markers of cell senescence. TAF frequencies were always higher in nfkb1 -/- mice ( ), reaching levels similar to those found in late-generation terc -/- mice ( ), a well-established model for telomere dysfunction-driven senescence. As expected for dysfunctional telomeres, chromatin bridges were observed between hepatocyte nuclei in nfkb1 -/- livers following partial hepatectomy ( ). To test whether the increase of TAF-positive cells was caused by inflammation rather than some other effect of p105/p50 deficiency, we first treated nfkb1 -/...
Feedback between inflammation and telomere dysfunction in vivo
However, telomeres are exquisitely sensitive to damage by ROS, generated extrinsically or intrinsically as a by-product of normal cellular metabolism. In cell senescence, ROS production is enhanced, generating DNA damage and increasing the DDR in a positive feed-forward loop. This loop is aggravated by telomere dysfunction, as seen in late generation terc -/- mice and in nfkb1 -/- cell senescence in vitro ( ). Accordingly, oxidative stress as measured by accumulation of 4-HNE, a marker for lipid peroxidation, was increased in nfkb1 -/- livers ( ) as well as broadband autofluorescence, another marker of oxidative damage ( ). 4-HNE-positive hepatocytes in nfkb1 -/- livers were also positive for the DNA damage- and senescence marker γH2AX ( ). Importantly, treatment of mice for 4 weeks with the antioxidant BHA rescued the TAF increase...
Methods
Experiments were performed on male nfkb1 -/- mice on a pure C57Bl/6 background and C57Bl/6 wt controls. Pure background mice were a gift from Jorge Caamano, (Birmingham University, UK). Experiments were performed at 36 weeks of age if not indicated otherwise. Immunohistochemistry was also performed on mixed C57Bl/6;129PF2/J nfkb1 -/- and F2 hybrid nfkb1 +/+ wt control mice (Jackson Laboratories, Bar Harbor, ME, USA) with identical results. p55 Δns/Δns mice and their wt littermate controls have been described. Male late-generation (F3-F4) terc -/- mice were bred from B6/Cg-TERC tm1Rdp /J (Jackson Laboratories). Lifespan studies were also performed in male C57Bl/6 under ad libitum feeding (AL) and under dietary restriction (DR, 60% of AL intake) and in male ICRFa (a long-lived substrain of C57Bl/6). Mice were housed in cages (56̴...
Ibuprofen and BHA treatment
For liver and gut regeneration studies, mice were given ibuprofen mixed in their food to a daily dosage of 50 mg per kg (mouse) per day. Treatment started at 8 weeks of age for 4 consecutive weeks. For senescence studies, mice received ibuprofen via pump (mini-osmotic pump, Alzet, model 2004) for a period of 8 weeks (starting at 24 weeks of age). Ibuprofen was dissolved in PEG and DMSO (50:50) to a daily dosage of 50 mg per kg. A small incision was made on the right flank and a mini pump was inserted subcutaneously and the wound was repaired with 7 mm clips. After 28 days a replacement was implanted. Under general anaesthesia, pumps were surgically removed and a wound repair was performed. A small incision was made on the left flank and a new mini pump was inserted subcutaneously and the wound was repaired with 7 mm clips. 8-week-old wt or nfkb1 -/-...
Measurement outputs
What raw and processed outputs should exist?
To understand the mechanisms by which chronic pro-inflammatory signals increased senescence-associated mitochondrial ROS production we first tested the role of p38MAPK. Signalli...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Mouse fibroblasts senesce spontaneously after few population doublings in 21% ambient oxygen as a consequence of a stress-induced DDR. Mouse adult ear fibroblasts (MAFs) from n...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
It is well established that the induction of the senescent phenotype including sen-β-Gal, SASP and enhanced ROS production requires at least 3-7 days following IR. M...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Telomere dysfunction is an important driver of cell senescence, and TAF ( ) are established as markers of telomere dysfunction and cell senescence in human and mouse tissues....
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Single comparisons were performed using two-tailed Student's t -test and multiple comparisons by one-way ANOVA followed by post hoc all pairwise multiple comparisons (Holm-Sidak).
from paperScoring or quantification
Quantify the primary readouts for this experiment: To understand the mechanisms by which chronic pro-inflammatory signals increased senescence-associated mitochondrial ROS production we first tested the role of p38MAPK. Signalli...; Mouse fibroblasts senesce spontaneously after few population doublings in 21% ambient oxygen as a consequence of a stress-induced DDR. Mouse adult ear fibroblasts (MAFs) from n...; It is well established that the induction of the senescent phenotype including sen-β-Gal, SASP and enhanced ROS production requires at least 3-7 days following IR. M...; Telomere dysfunction is an important driver of cell senescence, and TAF ( ) are established as markers of telomere dysfunction and cell senescence in human and mouse tissues.....
from paperStatistical comparison
Single comparisons were performed using two-tailed Student's t -test and multiple comparisons by one-way ANOVA followed by post hoc all pairwise multiple comparisons (Holm...
from paperReporting output
Report representative outputs alongside summary comparisons for To understand the mechanisms by which chronic pro-inflammatory signals increased senescence-associated mitochondrial ROS production we first tested the role of p38MAPK. Signalli..., Mouse fibroblasts senesce spontaneously after few population doublings in 21% ambient oxygen as a consequence of a stress-induced DDR. Mouse adult ear fibroblasts (MAFs) from n..., It is well established that the induction of the senescent phenotype including sen-β-Gal, SASP and enhanced ROS production requires at least 3-7 days following IR. M..., Telomere dysfunction is an important driver of cell senescence, and TAF ( ) are established as markers of telomere dysfunction and cell senescence in human and mouse tissues.....
inferred from protocolStructured statistical methods
Single comparisons were performed using two-tailed Student's t -test and multiple comparisons by one-way ANOVA followed by post hoc all pairwise multiple comparisons (Holm...
source structuredSource and audit
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Evidence quotes (8)
As chronic inflammation had been linked to ageing, we investigated whether nfkb1 -/- mice might age faster than their wt counterparts. We tested a wide range of biomarkers of mouse ageing in nfkb1 -/- mice at 36 weeks of age, their C57Bl/6 littermates at the same age and in old C57Bl/6 mice at 104 and 132 weeks ( ). With the exception of indicators of muscle weakness (ataxia, sarcopenia and kyphosis) and of spontaneous tumours, all tested biomarkers of ageing were more frequently positive in 36-week-old nfkb1 -/- mice as in their wt counterparts and often reached levels similar to wt mice aged ≥2 years ( ). Both mean and maximum lifespan were reduced in nfkb1 -/- mice ( ). At ~\n36 weeks of age, adult nfkb1 -/- mice showed frequent hair loss, early hair greying (starting at ~\n20 weeks of age), scruffy fur and cachexia, comparable to wt mice aged 104 weeks or older ( and ). In older nfkb1 -/- mice (ages above 44 weeks) massive hair loss, severe skin inflammation and delayed wound healing together with kyphosis (evidencing muscle loss) was occasionally seen ( ). Body mass curves in ageing mice show a t...
The gut epithelium is constantly regenerated from stem cells located at the bottom of the crypts. Low mucosal thickness of the colon and low villus length in the intestine are early morphological indicators of decreasing stem/progenitor function in the ageing gut, leading to malabsorption. Both parameters were decreased in nfkb1 -/- mice aged 36 weeks ( ) to values similar to those in 52-104-week-old wt mice. This was not associated with enhanced apoptosis ( ), suggesting a decreased regenerative capacity in either stem or progenitor cells. To address this, we isolated intestinal crypts from 12- and 54-week-old mice and analysed their growth in organotypic culture over a 10-day observation period ( ). We measured both the frequencies of crypts that started to grow, which is indicative of stem cell function, and growth rates, indicative of proliferation primarily in the progenitor cell compartment. Crypts isolated from young nfkb1 -/- mice maintained both the ability to grow ( ) and growth rates ( ) as well as wt crypts. In 54-week-old wt mice (~\n44% of median lifespan), the stem cell-related regenerative potential of intestinal crypts was comprom...
Mouse fibroblasts senesce spontaneously after few population doublings in 21% ambient oxygen as a consequence of a stress-induced DDR. Mouse adult ear fibroblasts (MAFs) from nfkb1 -/- donors senesced faster than their wt counterparts as indicated by accelerated loss of proliferative capacity ( ) and increased expression of the senescence marker senescence-associated β-galactosidase (sen-β-gal; ). To address the underlying mechanisms of accelerated senescence in nfkb1 -/- cells, we used a well-established model of induction of cell senescence by ionizing radiation (IR). Radiation-induced DDR can activate NF-κB through an ATM-dependent mechanism. However, the immediate response to DNA damage was not different between nfkb1 -/- and wt MAFs as shown by equal activation of ATM ( and ) and p53 ( ) and equal frequencies of DNA damage foci within 1 day after IR ( ). Similarly, there was no difference in the abundance of the NF-κB target COX-2 (PTGS-2) immediately after IR (, see also ).
It is well established that the induction of the senescent phenotype including sen-β-Gal, SASP and enhanced ROS production requires at least 3-7 days following IR. Multiple feedback loops interconnecting the DDR with SASP and ROS phenotypes via p38MAPK and NF-κB have been described, all of them stabilizing senescence with some kinetic delay. Accordingly, after a sufficient delay following IR, a wide range of senescence markers was elevated in both wt and nfkb1 -/- MAFs. Importantly, the senescent phenotype was aggravated in nfkb1 -/- cells according to every single marker tested ( and ). Specifically, there were more sen-β-Gal-positive cells ( ), more DNA damage foci per nucleus ( ), more mitochondrial superoxide was produced per cell ( ), more ROS accumulated in the cytoplasm ( ) and the expression of the CDKN1A and CDKN2A genes encoding the major cyclin-dependent kinase inhibitors p21 and p16 was more strongly upregulated in induced senescence in nfkb1 -/- MAFs ( ). The SASP was also stronger in nfkb1 -/- MAFs: A cytokine array confirmed enhanced secretion of 36 SASP components in induced senescence in wt M...
Telomere dysfunction is an important driver of cell senescence, and TAF ( ) are established as markers of telomere dysfunction and cell senescence in human and mouse tissues. TAF frequencies increased with age in liver hepatocytes and in enterocytes in intestinal crypts from wt mice ( ) similar to other markers of cell senescence. TAF frequencies were always higher in nfkb1 -/- mice ( ), reaching levels similar to those found in late-generation terc -/- mice ( ), a well-established model for telomere dysfunction-driven senescence. As expected for dysfunctional telomeres, chromatin bridges were observed between hepatocyte nuclei in nfkb1 -/- livers following partial hepatectomy ( ). To test whether the increase of TAF-positive cells was caused by inflammation rather than some other effect of p105/p50 deficiency, we first treated nfkb1 -/- mice for 8 weeks with the NSAID ibuprofen. Ibuprofen has complex, context-dependent effects on expression of pro-inflammatory cytokines, but robustly suppresses systemic COX activity. Enhanced TAF frequencies in nfkb1 -/- tissues were completely prevented by this treatment ( ). To...
However, telomeres are exquisitely sensitive to damage by ROS, generated extrinsically or intrinsically as a by-product of normal cellular metabolism. In cell senescence, ROS production is enhanced, generating DNA damage and increasing the DDR in a positive feed-forward loop. This loop is aggravated by telomere dysfunction, as seen in late generation terc -/- mice and in nfkb1 -/- cell senescence in vitro ( ). Accordingly, oxidative stress as measured by accumulation of 4-HNE, a marker for lipid peroxidation, was increased in nfkb1 -/- livers ( ) as well as broadband autofluorescence, another marker of oxidative damage ( ). 4-HNE-positive hepatocytes in nfkb1 -/- livers were also positive for the DNA damage- and senescence marker γH2AX ( ). Importantly, treatment of mice for 4 weeks with the antioxidant BHA rescued the TAF increase mediated by knockout of nfkb1 ( ). Additional markers of cell senescence including frequencies of γH2AX + PCNA - cells ( ), frequencies of ATM/ATR-positive cells ( ) and nuclear size ( ) were also enhanced in hepatocytes from ageing nfkb1 -/- mice. Moreover, there was a stron...
Experiments were performed on male nfkb1 -/- mice on a pure C57Bl/6 background and C57Bl/6 wt controls. Pure background mice were a gift from Jorge Caamano, (Birmingham University, UK). Experiments were performed at 36 weeks of age if not indicated otherwise. Immunohistochemistry was also performed on mixed C57Bl/6;129PF2/J nfkb1 -/- and F2 hybrid nfkb1 +/+ wt control mice (Jackson Laboratories, Bar Harbor, ME, USA) with identical results. p55 Δns/Δns mice and their wt littermate controls have been described. Male late-generation (F3-F4) terc -/- mice were bred from B6/Cg-TERC tm1Rdp /J (Jackson Laboratories). Lifespan studies were also performed in male C57Bl/6 under ad libitum feeding (AL) and under dietary restriction (DR, 60% of AL intake) and in male ICRFa (a long-lived substrain of C57Bl/6). Mice were housed in cages (56 cm × 38 cm × 18 cm, North Kent Plastics, Kent, UK) of groups of 4-6 that did not change from weaning. Mice were provided with saw dust and paper bedding and had AL access to water. Mice were housed at 20±2 °C under a 12 h light/12 h dark phot...
For liver and gut regeneration studies, mice were given ibuprofen mixed in their food to a daily dosage of 50 mg per kg (mouse) per day. Treatment started at 8 weeks of age for 4 consecutive weeks. For senescence studies, mice received ibuprofen via pump (mini-osmotic pump, Alzet, model 2004) for a period of 8 weeks (starting at 24 weeks of age). Ibuprofen was dissolved in PEG and DMSO (50:50) to a daily dosage of 50 mg per kg. A small incision was made on the right flank and a mini pump was inserted subcutaneously and the wound was repaired with 7 mm clips. After 28 days a replacement was implanted. Under general anaesthesia, pumps were surgically removed and a wound repair was performed. A small incision was made on the left flank and a new mini pump was inserted subcutaneously and the wound was repaired with 7 mm clips. 8-week-old wt or nfkb1 -/- mice were fed BHA (0.7% w/w) or normal chow for 4 weeks before undergoing partial hepatectomy.
Machine-readable layer
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"name": "Accelerated ageing in nfkb1 -/- mice",
"text": "As chronic inflammation had been linked to ageing, we investigated whether nfkb1 -/- mice might age faster than their wt counterparts. We tested a wide range of biomarkers of mouse ageing in nfkb1 -/- mice at 36 weeks of age, their C57Bl/6 littermates at the same age and in old C57Bl/6 mice at 104 and 132 weeks ( ). With the exception of indicators of muscle weakness (ataxia, sarcopenia and kyphosis) and of spontaneous tumours, all tested biomarkers of ageing were more frequently positive in 36-week-old nfkb1 -/- mice as in their wt counterparts and often reached levels similar to wt mice aged ≥2 years ( ). Both mean and maximum lifespan were reduced in nfkb1 -/- mice ( ). At ~\\n36 weeks of age, adult nfkb1 -/- mice showed frequent hair loss, early hair greying (starting at ~\\n20 weeks of age), scruffy fur and cachexia..."
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"text": "The gut epithelium is constantly regenerated from stem cells located at the bottom of the crypts. Low mucosal thickness of the colon and low villus length in the intestine are early morphological indicators of decreasing stem/progenitor function in the ageing gut, leading to malabsorption. Both parameters were decreased in nfkb1 -/- mice aged 36 weeks ( ) to values similar to those in 52-104-week-old wt mice. This was not associated with enhanced apoptosis ( ), suggesting a decreased regenerative capacity in either stem or progenitor cells. To address this, we isolated intestinal crypts from 12- and 54-week-old mice and analysed their growth in organotypic culture over a 10-day observation period ( ). We measured both the frequencies of crypts that started to grow, which is indicative of stem cell function, and growth rates, indicative of proliferation primarily in..."
},
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"name": "Chronic inflammation reinforces cellular senescence",
"text": "Mouse fibroblasts senesce spontaneously after few population doublings in 21% ambient oxygen as a consequence of a stress-induced DDR. Mouse adult ear fibroblasts (MAFs) from nfkb1 -/- donors senesced faster than their wt counterparts as indicated by accelerated loss of proliferative capacity ( ) and increased expression of the senescence marker senescence-associated β-galactosidase (sen-β-gal; ). To address the underlying mechanisms of accelerated senescence in nfkb1 -/- cells, we used a well-established model of induction of cell senescence by ionizing radiation (IR). Radiation-induced DDR can activate NF-κB through an ATM-dependent mechanism. However, the immediate response to DNA damage was not different between nfkb1 -/- and wt MAFs as shown by equal activation of ATM ( and ) and p53 ( ) and equal frequencies of DNA damage foc..."
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"name": "Chronic inflammation reinforces cellular senescence",
"text": "It is well established that the induction of the senescent phenotype including sen-β-Gal, SASP and enhanced ROS production requires at least 3-7 days following IR. Multiple feedback loops interconnecting the DDR with SASP and ROS phenotypes via p38MAPK and NF-κB have been described, all of them stabilizing senescence with some kinetic delay. Accordingly, after a sufficient delay following IR, a wide range of senescence markers was elevated in both wt and nfkb1 -/- MAFs. Importantly, the senescent phenotype was aggravated in nfkb1 -/- cells according to every single marker tested ( and ). Specifically, there were more sen-β-Gal-positive cells ( ), more DNA damage foci per nucleus ( ), more mitochondrial superoxide was produced per cell ( ), more ROS accumulated in the cytoplasm ( ) and the expression of the CDKN1A and CDKN2A genes encoding..."
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{
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"name": "Feedback between inflammation and telomere dysfunction in vivo",
"text": "Telomere dysfunction is an important driver of cell senescence, and TAF ( ) are established as markers of telomere dysfunction and cell senescence in human and mouse tissues. TAF frequencies increased with age in liver hepatocytes and in enterocytes in intestinal crypts from wt mice ( ) similar to other markers of cell senescence. TAF frequencies were always higher in nfkb1 -/- mice ( ), reaching levels similar to those found in late-generation terc -/- mice ( ), a well-established model for telomere dysfunction-driven senescence. As expected for dysfunctional telomeres, chromatin bridges were observed between hepatocyte nuclei in nfkb1 -/- livers following partial hepatectomy ( ). To test whether the increase of TAF-positive cells was caused by inflammation rather than some other effect of p105/p50 deficiency, we first treated nfkb1 -/\b..."
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"name": "Feedback between inflammation and telomere dysfunction in vivo",
"text": "However, telomeres are exquisitely sensitive to damage by ROS, generated extrinsically or intrinsically as a by-product of normal cellular metabolism. In cell senescence, ROS production is enhanced, generating DNA damage and increasing the DDR in a positive feed-forward loop. This loop is aggravated by telomere dysfunction, as seen in late generation terc -/- mice and in nfkb1 -/- cell senescence in vitro ( ). Accordingly, oxidative stress as measured by accumulation of 4-HNE, a marker for lipid peroxidation, was increased in nfkb1 -/- livers ( ) as well as broadband autofluorescence, another marker of oxidative damage ( ). 4-HNE-positive hepatocytes in nfkb1 -/- livers were also positive for the DNA damage- and senescence marker γH2AX ( ). Importantly, treatment of mice for 4 weeks with the antioxidant BHA rescued the TAF increase..."
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"name": "Methods",
"text": "Experiments were performed on male nfkb1 -/- mice on a pure C57Bl/6 background and C57Bl/6 wt controls. Pure background mice were a gift from Jorge Caamano, (Birmingham University, UK). Experiments were performed at 36 weeks of age if not indicated otherwise. Immunohistochemistry was also performed on mixed C57Bl/6;129PF2/J nfkb1 -/- and F2 hybrid nfkb1 +/+ wt control mice (Jackson Laboratories, Bar Harbor, ME, USA) with identical results. p55 Δns/Δns mice and their wt littermate controls have been described. Male late-generation (F3-F4) terc -/- mice were bred from B6/Cg-TERC tm1Rdp /J (Jackson Laboratories). Lifespan studies were also performed in male C57Bl/6 under ad libitum feeding (AL) and under dietary restriction (DR, 60% of AL intake) and in male ICRFa (a long-lived substrain of C57Bl/6). Mice were housed in cages (56̴..."
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"text": "For liver and gut regeneration studies, mice were given ibuprofen mixed in their food to a daily dosage of 50 mg per kg (mouse) per day. Treatment started at 8 weeks of age for 4 consecutive weeks. For senescence studies, mice received ibuprofen via pump (mini-osmotic pump, Alzet, model 2004) for a period of 8 weeks (starting at 24 weeks of age). Ibuprofen was dissolved in PEG and DMSO (50:50) to a daily dosage of 50 mg per kg. A small incision was made on the right flank and a mini pump was inserted subcutaneously and the wound was repaired with 7 mm clips. After 28 days a replacement was implanted. Under general anaesthesia, pumps were surgically removed and a wound repair was performed. A small incision was made on the left flank and a new mini pump was inserted subcutaneously and the wound was repaired with 7 mm clips. 8-week-old wt or nfkb1 -/-..."
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