Changes in gut, microbiome, and cognition after doxorubicin, cyclophosphamide, and paclitaxel chemotherapy treatment methods
Aim. Evidence-backed execution summary for Changes in gut, microbiome, and cognition after doxorubicin, cyclophosphamide, and paclitaxel chemotherapy treatment methods from Changes in gut, microbiome, and cognition after doxorubicin, cyclophosphamide, and paclitaxel chemotherapy treatment.
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This experiment, in seven questions
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Shopping and prep list
What do I need before I start?
mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Animals
reagent used in the protocol.
- Use
- Twelve-week-old female C57/BL6J mice were purchased from Jackson Laboratory. Female mice were used as this was a breast cancer drug paradigm, and diagnosis of breast cancer in males makes up less than 1% of all new cancer diagnoses. Mice were housed 4 per cage in a climate-controlled facility on a 12 h light/dark c...
Drug paradigm
reagent used in the protocol.
- Use
- Chemotherapeutic agents were purchased from the UAMS Inpatient Pharmacy, diluted into stock solutions and stored according to the manufacturer's instructions. Chemotherapeutic agent dosages used were previously shown by our lab to induce hippocampal morphological and behavioral changes while being translatable...
Drug paradigm
reagent used in the protocol.
- Use
- Fig. 1 Experimental timeline. figure generated with BioRender.com.
CME bHPLC TMT Methods - Orbitrap eclipse
reagent used in the protocol.
- Use
- Each super-fraction was then further separated by reverse phase XSelect CSH C18 2.5 µm resin (Waters) on an in-line 150 × 0.075 mm column using an UltiMate 3000 RSLCnano system (Thermo). Peptides were eluted using a 75 min gradient from 98:2 to 60:40 buffer A: B ratio. Eluted pep...
RNA sequencing (hippocampal tissue)
reagent used in the protocol.
- Use
- After behavior testing, whole brains were harvested and flash frozen in 2-methylbutane. Hippocampal samples were collected from coronal slices using a 0.2 mm tissue punch and were sent to the RNA Sequencing Core (UAMS, Little Rock, AR) for processing ( n = 5-6). RNA was extracted from samples using the Zymo Qu...
Western blot (hippocampal tissue)
reagent used in the protocol.
- Use
- A Potter-Elvehjem mechanical compact stirrer (BDC2002, Caframo LabSolutions), was used to homogenize frozen hippocampal samples in a 1% Triton-X100 radioimmunoprecipitation buffer (MilliporeSigma) containing protease inhibitors. Bicinchoninic acid (BCA) protein assay (Bio-Rad) was used to determine the protein conce...
Proteomics (intestinal tissue)
reagent used in the protocol.
- Use
- Intestines were homogenized in RIPA buffer on ice. Homogenates were centrifuged and supernatants collected. Protein concentrations were determined with the Bradford assay kit (Thermo Fisher) following the manufacturer's instructions.
CME bHPLC TMT Methods - Orbitrap eclipse
reagent used in the protocol.
- Use
- Total protein from each sample was reduced, alkylated, and purified by chloroform/methanol extraction prior to digestion with sequencing grade trypsin (Promega). The resulting peptides were labeled using a tandem mass tag 10-plex isobaric label reagent set (Thermo) and combined into one multiplex sample group. Label...
RNA Sequencing- IPA networks: AC-T disrupts nervous system development
Associated network functions: Embryonic Development, Nervous System Development and Function, Organ Development
- Use
- Associated network functions: Embryonic Development, Nervous System Development and Function, Organ Development
RNA Sequencing- IPA networks: AC-T disrupts nervous system development
Number of "focus molecules" contained in network: 28
- Use
- Number of "focus molecules" contained in network: 28
RNA Sequencing- IPA networks: AC-T disrupts nervous system development
Network molecules: ACAA1, ADAMTS15, ADAMTS18, ADAMTS19, ALOX12B, ALPK2, angiotensin II receptor type 1, BCL11A, CNTNAP3, Collagen type IV, CPS1, DHRS2, DKKL1, DNA-PK, DTL, EBF2, EBF3, ECM, EGLN, ERFE, FAM83F, HBA2, LRIG1, MAN1A1, MATN2, Mup1 (includes others), PADI1, POFUT2 substrates, QPRT, RPRM, VASH2, VEGF, ZIC1,...
- Use
- Network molecules: ACAA1, ADAMTS15, ADAMTS18, ADAMTS19, ALOX12B, ALPK2, angiotensin II receptor type 1, BCL11A, CNTNAP3, Collagen type IV, CPS1, DHRS2, DKKL1, DNA-PK, DTL, EBF2, EBF3, ECM, EGLN, ERFE, FAM83F, HBA2, LRIG1, MAN1A1, MATN2, Mup1 (includes others), PADI1, POFUT2 substrates, QPRT, RPRM, VASH2, VEGF, ZIC1,...
RNA Sequencing- IPA networks: AC-T disrupts nervous system development
Associated network functions: Cellular Development, Cellular Growth and Proliferation, Nervous System Development and Function
- Use
- Associated network functions: Cellular Development, Cellular Growth and Proliferation, Nervous System Development and Function
RNA Sequencing- IPA networks: AC-T disrupts nervous system development
Number of "focus molecules" contained in network: 27
- Use
- Number of "focus molecules" contained in network: 27
RNA Sequencing- IPA networks: AC-T disrupts nervous system development
Network molecules: ACHR alpha, C1QL4, CALB2, CHRNA10, CHRNA3, CHRNA5, CIAP, DRGX, EDARADD, GBX2, GJC1, GLT8D2, HTR3A, Htr5b, Inflammasome, IRX1, IRX2, IRX3, IRX4, IRX5, ISL1, MAB21L1, NFkB (complex), NLR, NLRC3, NOD2, NRG (family), P2RX5, PIRT, PLC beta, POU4F1, serotonin receptor, SLC3A1, SLC41A3, TRIM40
- Use
- Network molecules: ACHR alpha, C1QL4, CALB2, CHRNA10, CHRNA3, CHRNA5, CIAP, DRGX, EDARADD, GBX2, GJC1, GLT8D2, HTR3A, Htr5b, Inflammasome, IRX1, IRX2, IRX3, IRX4, IRX5, ISL1, MAB21L1, NFkB (complex), NLR, NLRC3, NOD2, NRG (family), P2RX5, PIRT, PLC beta, POU4F1, serotonin receptor, SLC3A1, SLC41A3, TRIM40
RNA Sequencing- IPA networks: AC-T disrupts nervous system development
Associated network functions: Cellular Development, Connective Tissue Disorders, Nervous System Development and Function
- Use
- Associated network functions: Cellular Development, Connective Tissue Disorders, Nervous System Development and Function
RNA Sequencing- IPA networks: AC-T disrupts nervous system development
Number of "focus molecules" contained in network: 26
- Use
- Number of "focus molecules" contained in network: 26
Data analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Proteins were identified and MS3 reporter ions quantified using MaxQuant (Max Planck Institute) against the UniprotKB Mus musculus (March 2021) database with a parent ion tolerance of 3 ppm, a fragment ion tolerance of 0.5 Da, and a reporter ion tolerance of 0.003 Da. Scaffold Q + S (Proteome Software) was used to v...
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Open quote workflowStep-by-step procedure
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Drug paradigm
Chemotherapeutic agents were purchased from the UAMS Inpatient Pharmacy, diluted into stock solutions and stored according to the manufacturer's instructions. Chemotherapeutic agent dosages used were previously shown by our lab to induce hippocampal morphological and behavioral changes while being translatable to human dosages. DOX (A) was administered via intraperitoneal (I.P.) injection at 2 mg/kg dosage in combination with CYP (C) at 50 mg/kg constituting the AC chemotherapy regimen. AC was given once a week for four weeks. Upon completion of the AC injections, PTX (5 mg/kg) was administered once weekly via IP injections for four weeks, completing the AC-T regimen. Control mice received 0.9% saline for all injections. Behavior testing began 30 days following the last injection, and euthanasia by cervical dislocation occurred 30 min after the last behavior test. Experimental...
Behavior testing- sociability
The three-chamber sociability test is depicted below (Fig. ),. Briefly, a polycarbonate box was divided into three chambers with removable partitions that had openings to allow free movement throughout the chambers. During the habituation stage, the test mouse was placed in the center chamber and was allowed to freely explore for 10 min (Stage 1). After 10 min, a mouse of similar age, sex, and strain (Stranger 1), was placed inside a small wire cage in one of the side chambers (Stage 2). The test mouse was allowed to familiarize itself with Stranger 1 for 10 min before a second stranger (Stranger 2) was placed in the previously empty wire cage in the other side chamber (Stage 3). The test mouse was again allowed to explore Stranger 2 for 10 min (Social Novelty phase). Data analyzed compared the percentage of time spent with Stranger 1 versus Stranger 2. Location of Stranger 1 versus...
Open-Field (OF) test
To assess the unconditioned anxiety of mice the OF behavioral test was employed (Fig. ). The mice were placed in the middle of a square Plexiglass arena (40 cm x 40 cm x 35 cm) with a gray floor. The walls of the arena were covered with white posterboard to prevent the mouse from seeing the room's surroundings. The mouse was allowed to roam freely for 10 min. At the end of the trial, the number of feces were counted. The arena was cleaned with 20% ethanol between animals to minimize olfactory signals between trials. Locomotor activity was measured using total distance traveled (cm) and velocity (cm/s). The center region of the arena (20 cm x 20 cm) is considered anxiogenic. Time spent in the center zone was recorded.
RNA sequencing (hippocampal tissue)
After behavior testing, whole brains were harvested and flash frozen in 2-methylbutane. Hippocampal samples were collected from coronal slices using a 0.2 mm tissue punch and were sent to the RNA Sequencing Core (UAMS, Little Rock, AR) for processing ( n = 5-6). RNA was extracted from samples using the Zymo Quick-DNA/RNA miniprep Plus Kit (Cat # D7003). mRNA Library was constructed using the Illumina TruSeq Stranded mRNA Library Prep kit (Cat # 20020594). Samples were run on a 200-cycle NextSeq 2000 P2 flow cell. Dragen (v 4.3.6) was used for binary base call conversion and FastQC (v 0.11.9) was used for quality control. Quantification was performed using Salmon (v 1.9.0). Fastq files were uploaded into Qiagen Ingenuity Pathway Analysis (IPA) via the RNA-Seq Analysis Portal.). All analysis was performed using an FDR p-value < 0.05, and a fold change > 1.5 or < -1.5.
CME bHPLC TMT Methods - Orbitrap eclipse
Total protein from each sample was reduced, alkylated, and purified by chloroform/methanol extraction prior to digestion with sequencing grade trypsin (Promega). The resulting peptides were labeled using a tandem mass tag 10-plex isobaric label reagent set (Thermo) and combined into one multiplex sample group. Labeled peptides were separated into 46 fractions on a 100 × 1.0 mm Acquity BEH C18 column (Waters) using an UltiMate 3000 UHPLC system (Thermo) with a 50 min gradient from 99:1 to 60:40 buffer A: B ratio under basic pH conditions, then consolidated into 18 super-fractions. Buffer A = 10 mM ammonium hydroxide, 0.5% acetonitrile, Buffer B = 10 mM ammonium hydroxide, 99.9% acetonitrile, both buffers adjusted to pH 10 for offline separation.
Behavior- open field: AC-T increases anxiety-like behavior
Anxiogenic behavior in the OF test is measured by a parameter known as central tendency, or the amount of time each mouse spends in the center of the arena. Central tendency was used to measure anxiogenic behavior in the OF test. Decreased central tendency indicates increased anxiety. There was a significant decrease in central tendency between the saline and AC-T groups (Fig. ) ( p = 0.0349).
Differential analysis of protein expression: AC-T alters IL-15 signaling in intestinal tissue
Ingenuity Pathway Analysis tool was used to analyze proteomics networks affected by the treatment regimen in intestinal tissue. Qiagen IPA algorithm overlays focus molecules from the experimental dataset to the Global Molecular Network and generates a connectivity map. The p -score (-log 10 (p-value)) is calculated by Fisher's exact test and is indicative of the probability of focus molecules in a network being selected randomly from the Global Molecular Network. Table Fig. contains the top 4 networks.
Crypt depth and villus height: AC-T significantly decreases crypt depth
The intestinal derangement was observed 30 days after AC-T treatment. When the mean depth of crypts in mice treated with (145.00 ± 6.23) AC-T was compared to the (125.6 ± 2.44) saline group, a significant decrease in average depth was observed (t = 3.042, p < 0.01; Fig. ). Villus length in mice treated with (326.7 ± 14.94 µm) AC-T was not significantly affected compared to (305.3 ± 7.66 µm) saline (t = 1.287, p = 0.20; Fig. ).
Measurement outputs
What raw and processed outputs should exist?
Network proteins: ARHGEF17, ASCL2, BRCA1, CHUK, COG6, COL17A1, CSRP1, CTNNB1, Cops2, Crip2, EGFR, EVX1, H1-5, H3-3 A/H3-3B, HPF1, KIAA1217, MAL2, MRM2, MRPS27, MYC, MYL9, 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
Associated network functions: Molecular Transport, Cell Signaling, Vitamin and Mineral Metabolism
- 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
Number of "focus molecules" contained in network: 9
- 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
Network proteins: Agtr1b, Androgen-AR, C1orf50, CBL, CCL3L1, CLCA4, CYP27B1, Ca2+, Calbindin, DDIAS, ENPEP, H6PD, HTN3, IFNG, IL1B, KLK1, LYZ, MCFD2, PER3, PTHLH, PTK2, Pdlim3,...
- 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
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Proteins were identified and MS3 reporter ions quantified using MaxQuant (Max Planck Institute) against the UniprotKB Mus musculus (March 2021) database with a parent ion tolerance of 3 ppm, a fragment ion tolerance of 0.5 Da, and a reporter ion tolerance of 0.003 Da.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Network proteins: ARHGEF17, ASCL2, BRCA1, CHUK, COG6, COL17A1, CSRP1, CTNNB1, Cops2, Crip2, EGFR, EVX1, H1-5, H3-3 A/H3-3B, HPF1, KIAA1217, MAL2, MRM2, MRPS27, MYC, MYL9, N...; Associated network functions: Molecular Transport, Cell Signaling, Vitamin and Mineral Metabolism; Number of "focus molecules" contained in network: 9; Network proteins: Agtr1b, Androgen-AR, C1orf50, CBL, CCL3L1, CLCA4, CYP27B1, Ca2+, Calbindin, DDIAS, ENPEP, H6PD, HTN3, IFNG, IL1B, KLK1, LYZ, MCFD2, PER3, PTHLH, PTK2, Pdlim3,....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Proteins were identified and MS3 reporter ions quantified using MaxQuant (Max Planck Institute) against the UniprotKB Mus musculus (March 2021) database with a parent ion tolera...; Various alpha diversity metrics (within sample diversity) were estimated using various rarefaction approaches and samples were rarefied approximately 8800-10,000 sequences per s...; Between sample diversity was estimated using UniFrac (weighted and unweighted) and Bray-Curtis dissimilarity using q2-diversity after rarefying samples to 8898 sequences per sam...; ANCOM-BC was used, via q2-composition, to estimate the differential abundance of non-rarefied features, which were subsequently collapsed by taxonomy at the genus level. Only si...
from paperReporting output
Report representative outputs alongside summary comparisons for Network proteins: ARHGEF17, ASCL2, BRCA1, CHUK, COG6, COL17A1, CSRP1, CTNNB1, Cops2, Crip2, EGFR, EVX1, H1-5, H3-3 A/H3-3B, HPF1, KIAA1217, MAL2, MRM2, MRPS27, MYC, MYL9, N..., Associated network functions: Molecular Transport, Cell Signaling, Vitamin and Mineral Metabolism, Number of "focus molecules" contained in network: 9, Network proteins: Agtr1b, Androgen-AR, C1orf50, CBL, CCL3L1, CLCA4, CYP27B1, Ca2+, Calbindin, DDIAS, ENPEP, H6PD, HTN3, IFNG, IL1B, KLK1, LYZ, MCFD2, PER3, PTHLH, PTK2, Pdlim3,....
inferred from protocolStructured statistical methods
Proteins were identified and MS3 reporter ions quantified using MaxQuant (Max Planck Institute) against the UniprotKB Mus musculus (March 2021) database with a parent ion tolera...; Various alpha diversity metrics (within sample diversity) were estimated using various rarefaction approaches and samples were rarefied approximately 8800-10,000 sequences per s...; Between sample diversity was estimated using UniFrac (weighted and unweighted) and Bray-Curtis dissimilarity using q2-diversity after rarefying samples to 8898 sequences per sam...; ANCOM-BC was used, via q2-composition, to estimate the differential abundance of non-rarefied features, which were subsequently collapsed by taxonomy at the genus level. Only si...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Chemotherapeutic agents were purchased from the UAMS Inpatient Pharmacy, diluted into stock solutions and stored according to the manufacturer's instructions. Chemotherapeutic agent dosages used were previously shown by our lab to induce hippocampal morphological and behavioral changes while being translatable to human dosages. DOX (A) was administered via intraperitoneal (I.P.) injection at 2 mg/kg dosage in combination with CYP (C) at 50 mg/kg constituting the AC chemotherapy regimen. AC was given once a week for four weeks. Upon completion of the AC injections, PTX (5 mg/kg) was administered once weekly via IP injections for four weeks, completing the AC-T regimen. Control mice received 0.9% saline for all injections. Behavior testing began 30 days following the last injection, and euthanasia by cervical dislocation occurred 30 min after the last behavior test. Experimental timeline is represented in Fig..
The three-chamber sociability test is depicted below (Fig. ),. Briefly, a polycarbonate box was divided into three chambers with removable partitions that had openings to allow free movement throughout the chambers. During the habituation stage, the test mouse was placed in the center chamber and was allowed to freely explore for 10 min (Stage 1). After 10 min, a mouse of similar age, sex, and strain (Stranger 1), was placed inside a small wire cage in one of the side chambers (Stage 2). The test mouse was allowed to familiarize itself with Stranger 1 for 10 min before a second stranger (Stranger 2) was placed in the previously empty wire cage in the other side chamber (Stage 3). The test mouse was again allowed to explore Stranger 2 for 10 min (Social Novelty phase). Data analyzed compared the percentage of time spent with Stranger 1 versus Stranger 2. Location of Stranger 1 versus Stranger 2 was appropriately counterbalanced. The apparatus was cleaned with 20% ethanol and allowed to air dry before the next trial began. Behavior was recorded and tracked using the Ethovision tracking system for all behavior sessions (Ethovision XT v17.5, Noldus Information Technology, Wagenin...
To assess the unconditioned anxiety of mice the OF behavioral test was employed (Fig. ). The mice were placed in the middle of a square Plexiglass arena (40 cm x 40 cm x 35 cm) with a gray floor. The walls of the arena were covered with white posterboard to prevent the mouse from seeing the room's surroundings. The mouse was allowed to roam freely for 10 min. At the end of the trial, the number of feces were counted. The arena was cleaned with 20% ethanol between animals to minimize olfactory signals between trials. Locomotor activity was measured using total distance traveled (cm) and velocity (cm/s). The center region of the arena (20 cm x 20 cm) is considered anxiogenic. Time spent in the center zone was recorded.
After behavior testing, whole brains were harvested and flash frozen in 2-methylbutane. Hippocampal samples were collected from coronal slices using a 0.2 mm tissue punch and were sent to the RNA Sequencing Core (UAMS, Little Rock, AR) for processing ( n = 5-6). RNA was extracted from samples using the Zymo Quick-DNA/RNA miniprep Plus Kit (Cat # D7003). mRNA Library was constructed using the Illumina TruSeq Stranded mRNA Library Prep kit (Cat # 20020594). Samples were run on a 200-cycle NextSeq 2000 P2 flow cell. Dragen (v 4.3.6) was used for binary base call conversion and FastQC (v 0.11.9) was used for quality control. Quantification was performed using Salmon (v 1.9.0). Fastq files were uploaded into Qiagen Ingenuity Pathway Analysis (IPA) via the RNA-Seq Analysis Portal.). All analysis was performed using an FDR p-value < 0.05, and a fold change > 1.5 or < -1.5.
Total protein from each sample was reduced, alkylated, and purified by chloroform/methanol extraction prior to digestion with sequencing grade trypsin (Promega). The resulting peptides were labeled using a tandem mass tag 10-plex isobaric label reagent set (Thermo) and combined into one multiplex sample group. Labeled peptides were separated into 46 fractions on a 100 × 1.0 mm Acquity BEH C18 column (Waters) using an UltiMate 3000 UHPLC system (Thermo) with a 50 min gradient from 99:1 to 60:40 buffer A: B ratio under basic pH conditions, then consolidated into 18 super-fractions. Buffer A = 10 mM ammonium hydroxide, 0.5% acetonitrile, Buffer B = 10 mM ammonium hydroxide, 99.9% acetonitrile, both buffers adjusted to pH 10 for offline separation.
Anxiogenic behavior in the OF test is measured by a parameter known as central tendency, or the amount of time each mouse spends in the center of the arena. Central tendency was used to measure anxiogenic behavior in the OF test. Decreased central tendency indicates increased anxiety. There was a significant decrease in central tendency between the saline and AC-T groups (Fig. ) ( p = 0.0349).
Ingenuity Pathway Analysis tool was used to analyze proteomics networks affected by the treatment regimen in intestinal tissue. Qiagen IPA algorithm overlays focus molecules from the experimental dataset to the Global Molecular Network and generates a connectivity map. The p -score (-log 10 (p-value)) is calculated by Fisher's exact test and is indicative of the probability of focus molecules in a network being selected randomly from the Global Molecular Network. Table Fig. contains the top 4 networks.
The intestinal derangement was observed 30 days after AC-T treatment. When the mean depth of crypts in mice treated with (145.00 ± 6.23) AC-T was compared to the (125.6 ± 2.44) saline group, a significant decrease in average depth was observed (t = 3.042, p < 0.01; Fig. ). Villus length in mice treated with (326.7 ± 14.94 µm) AC-T was not significantly affected compared to (305.3 ± 7.66 µm) saline (t = 1.287, p = 0.20; Fig. ).
Machine-readable layer
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"name": "Changes in gut, microbiome, and cognition after doxorubicin, cyclophosphamide, and paclitaxel chemotherapy treatment methods",
"description": "Evidence-backed execution summary for Changes in gut, microbiome, and cognition after doxorubicin, cyclophosphamide, and paclitaxel chemotherapy treatment methods from Changes in gut, microbiome, and cognition after doxorubicin, cyclophosphamide, and paclitaxel chemotherapy treatment.",
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"name": "Drug paradigm",
"text": "Chemotherapeutic agents were purchased from the UAMS Inpatient Pharmacy, diluted into stock solutions and stored according to the manufacturer's instructions. Chemotherapeutic agent dosages used were previously shown by our lab to induce hippocampal morphological and behavioral changes while being translatable to human dosages. DOX (A) was administered via intraperitoneal (I.P.) injection at 2 mg/kg dosage in combination with CYP (C) at 50 mg/kg constituting the AC chemotherapy regimen. AC was given once a week for four weeks. Upon completion of the AC injections, PTX (5 mg/kg) was administered once weekly via IP injections for four weeks, completing the AC-T regimen. Control mice received 0.9% saline for all injections. Behavior testing began 30 days following the last injection, and euthanasia by cervical dislocation occurred 30 min after the last behavior test. Experimental..."
},
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"@type": "HowToStep",
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"name": "Behavior testing- sociability",
"text": "The three-chamber sociability test is depicted below (Fig. ),. Briefly, a polycarbonate box was divided into three chambers with removable partitions that had openings to allow free movement throughout the chambers. During the habituation stage, the test mouse was placed in the center chamber and was allowed to freely explore for 10 min (Stage 1). After 10 min, a mouse of similar age, sex, and strain (Stranger 1), was placed inside a small wire cage in one of the side chambers (Stage 2). The test mouse was allowed to familiarize itself with Stranger 1 for 10 min before a second stranger (Stranger 2) was placed in the previously empty wire cage in the other side chamber (Stage 3). The test mouse was again allowed to explore Stranger 2 for 10 min (Social Novelty phase). Data analyzed compared the percentage of time spent with Stranger 1 versus Stranger 2. Location of Stranger 1 versus..."
},
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"@type": "HowToStep",
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"name": "Open-Field (OF) test",
"text": "To assess the unconditioned anxiety of mice the OF behavioral test was employed (Fig. ). The mice were placed in the middle of a square Plexiglass arena (40 cm x 40 cm x 35 cm) with a gray floor. The walls of the arena were covered with white posterboard to prevent the mouse from seeing the room's surroundings. The mouse was allowed to roam freely for 10 min. At the end of the trial, the number of feces were counted. The arena was cleaned with 20% ethanol between animals to minimize olfactory signals between trials. Locomotor activity was measured using total distance traveled (cm) and velocity (cm/s). The center region of the arena (20 cm x 20 cm) is considered anxiogenic. Time spent in the center zone was recorded."
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"text": "After behavior testing, whole brains were harvested and flash frozen in 2-methylbutane. Hippocampal samples were collected from coronal slices using a 0.2 mm tissue punch and were sent to the RNA Sequencing Core (UAMS, Little Rock, AR) for processing ( n = 5-6). RNA was extracted from samples using the Zymo Quick-DNA/RNA miniprep Plus Kit (Cat # D7003). mRNA Library was constructed using the Illumina TruSeq Stranded mRNA Library Prep kit (Cat # 20020594). Samples were run on a 200-cycle NextSeq 2000 P2 flow cell. Dragen (v 4.3.6) was used for binary base call conversion and FastQC (v 0.11.9) was used for quality control. Quantification was performed using Salmon (v 1.9.0). Fastq files were uploaded into Qiagen Ingenuity Pathway Analysis (IPA) via the RNA-Seq Analysis Portal.). All analysis was performed using an FDR p-value < 0.05, and a fold change > 1.5 or < -1.5."
},
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"name": "CME bHPLC TMT Methods - Orbitrap eclipse",
"text": "Total protein from each sample was reduced, alkylated, and purified by chloroform/methanol extraction prior to digestion with sequencing grade trypsin (Promega). The resulting peptides were labeled using a tandem mass tag 10-plex isobaric label reagent set (Thermo) and combined into one multiplex sample group. Labeled peptides were separated into 46 fractions on a 100 × 1.0 mm Acquity BEH C18 column (Waters) using an UltiMate 3000 UHPLC system (Thermo) with a 50 min gradient from 99:1 to 60:40 buffer A: B ratio under basic pH conditions, then consolidated into 18 super-fractions. Buffer A = 10 mM ammonium hydroxide, 0.5% acetonitrile, Buffer B = 10 mM ammonium hydroxide, 99.9% acetonitrile, both buffers adjusted to pH 10 for offline separation."
},
{
"@type": "HowToStep",
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"name": "Behavior- open field: AC-T increases anxiety-like behavior",
"text": "Anxiogenic behavior in the OF test is measured by a parameter known as central tendency, or the amount of time each mouse spends in the center of the arena. Central tendency was used to measure anxiogenic behavior in the OF test. Decreased central tendency indicates increased anxiety. There was a significant decrease in central tendency between the saline and AC-T groups (Fig. ) ( p = 0.0349)."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Differential analysis of protein expression: AC-T alters IL-15 signaling in intestinal tissue",
"text": "Ingenuity Pathway Analysis tool was used to analyze proteomics networks affected by the treatment regimen in intestinal tissue. Qiagen IPA algorithm overlays focus molecules from the experimental dataset to the Global Molecular Network and generates a connectivity map. The p -score (-log 10 (p-value)) is calculated by Fisher's exact test and is indicative of the probability of focus molecules in a network being selected randomly from the Global Molecular Network. Table Fig. contains the top 4 networks."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Crypt depth and villus height: AC-T significantly decreases crypt depth",
"text": "The intestinal derangement was observed 30 days after AC-T treatment. When the mean depth of crypts in mice treated with (145.00 ± 6.23) AC-T was compared to the (125.6 ± 2.44) saline group, a significant decrease in average depth was observed (t = 3.042, p < 0.01; Fig. ). Villus length in mice treated with (326.7 ± 14.94 µm) AC-T was not significantly affected compared to (305.3 ± 7.66 µm) saline (t = 1.287, p = 0.20; Fig. )."
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