Changes in gut, microbiome, and cognition after doxorubicin, cyclophosphamide, and paclitaxel chemotherapy treatment methods
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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. ).
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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. ).
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