Continuous, Automated Breathing Rate and Body Motion Monitoring of Rats With Paraquat-Induced Progressive Lung Injury

Abstract

Assessments of respiratory response and animal activity are useful endpoints in drug pharmacology and safety research. We investigated whether continuous, direct monitoring of breathing rate and body motion in animals in the home cage using the Vum Digital Smart House can complement standard measurements in enabling more granular detection of the onset and severity of physiologic events related to lung injury in a well-established rodent model of paraquat (PQ) toxicity. In rats administered PQ, breathing rate was significantly elevated while body motion was significantly reduced following dosing and extending throughout the 14-day study duration for breathing rate and at least 5 days for both nighttime and daytime body motion. Time course differences in these endpoints in response to the potential ameliorative test article bardoxolone were also readily detected. More complete than standard in-life measurements, breathing rate and body motion tracked injury progression continuously over the full study time period and aligned with, and informed on interval changes in clinical pathology. In addition, breathing rates correlated with terminal pathology measurements, such as normalized lung weights and histologic alveolar damage and edema. This study is a preliminary evaluation of the technology; our results demonstrate that continuously measured breathing rate and body motion served as physiologically relevant readouts to assess lung injury progression and drug response in a respiratory injury animal model.

Keywords: activity; breathing rate; digital biomarkers; drug discovery; lung injury; paraquat; rodent; translational research.

Figure 1

Standard in-life measurements confirmed lung injury and detected effects of bardoxolone treatment in a rodent model of paraquat (PQ)-induced lung injury (PQiLI). (A) Experimental timeline. After a 14-day acclimation period, rats were given an intra-tracheal dose of PQ on Day 0. Rats were orally treated with bardoxolone or vehicle either prophylactically starting the day before PQ administration (Day 1) or therapeutically starting the day after PQ administration (Day 1) daily until study end. Treatment schedules are shown in brackets. Three separate subgroups of rats per study group were euthanized on either Days 3, 6, or 14 for endpoint tissue collection. Automated breathing rate and body motion readouts were collected continuously throughout the study, and standard in-life measurements (body weight and body temperature) were collected daily during the study. (B) Change in body weight. Compared with control rats (SA/Veh/P), body weight was decreased in PQ/Veh/P and PQ/Veh/T rats. These changes were observed as early as Day 1 and as late as Day 14. Bardoxolone-treated rats (PQ/Bar/P and PQ/Bar/T) showed lower body weights compared with vehicle-treated counterparts. ^*p ≤ 0.05 vs. SA/Veh/P. ^ap ≤ 0.01 PQ/Veh/P vs. PQ/Bar/P, and ^bp ≤ 0.01 PQ/Veh/T vs. PQ/Bar/T. (C) Change in body temperature. Compared with SA/Veh/P rats, body temperature was decreased in PQ/Veh/P and PQ/Veh/T rats as early as Day 1 and as late as Day 3. Despite showing an earlier decrease in body temperature, PQ/Bar/T rats demonstrated higher body temperatures on Days 6–8 and Day 10. (B¹,C¹) Day 3 (n = 24 rats for all study groups except n = 23 for SA/Veh/P). (B²,C²) Day 6 (n = 16 rats for all study groups). (B³,C³) Day 14 (n = 8 rats for all study groups except n = 7 for SA/Veh/P). ^*p ≤ 0.05 vs. SA/Veh/P. ^ap ≤ 0.05 PQ/Veh/P vs. PQ/Bar/P and ^bp ≤ 0.05 PQ/Veh/T vs. PQ/Bar/T. Error bars are +/− SEM.

Figure 2

Automated breathing rate and body motion detected injury and effects of bardoxolone in a rodent model of PQiLI. (A) Change in breathing rate. Compared with control rats (SA/Veh/P), breathing rate was increased in PQ/Veh/P and PQ/Veh/T rats. Elevated breathing rate was observed as early as Day 1 and as late as Day 14. PQ/Bar/P, but not PQ/Bar/T, rats had lower breathing rates compared with vehicle-treated counterparts. ^*p ≤ 0.01 vs. SA/Veh/P, ^ap ≤ 0.05 PQ/Veh/P vs. PQ/Bar/P, and ^bp ≤ 0.05 PQ/Veh/T vs. PQ/Bar/T. (B) Change in nighttime body motion. Compared with SA/Veh/P rats, nighttime body motion was decreased in PQ/Veh/P and PQ/Veh/T rats as early as Day 1 and as late as Day 5. The recovery of nighttime body motion in PQ/Bar/P and PQ/Bar/T rats was delayed. ^*p ≤ 0.01 vs. SA/Veh/P. ^ap ≤ 0.05 PQ/Veh/P vs. PQ/Bar/P and ^bp ≤ 0.01 PQ/Veh/T vs. PQ/Bar/T. (C) Change in daytime body motion. Compared with SA/Veh/P rats, daytime body motion was decreased in PQ/Veh/P and PQ/Veh/T rats as early as Day 1 and as late as Day 12. PQ/Bar/P and PQ/Bar/T rats showed lower daytime body motion compared with vehicle-treated counterparts during specific days. (A¹,B¹,C¹) Day 3 (n = 24 rats for all study groups except n = 23 for SA/Veh/P). (A²,B²,C²) Day 6 (n = 16 rats for all study groups). (A³,B³,C³) Day 14 (n = 8 rats for all study groups except n = 7 for SA/Veh/P). ^*p ≤ 0.05 vs. SA/Veh/P. ^ap ≤ 0.05 PQ/Veh/P vs. PQ/Bar/P and ^bp ≤ 0.05 PQ/Veh/T vs. PQ/Bar/T. Error bars are +/− SEM.

Figure 3

Clinical pathology performed at distinct time points showed changes in response to PQ administration and bardoxolone treatment. (A) Triglycerides. PQ/Veh/P rats showed lower triglyceride levels on Day 3 compared with SA/Veh/P. Bardoxolone-treated rats (PQ/Bar/P and PQ/Bar/T) demonstrated changes as early as Day 3 and as late as Day 14. (B) Alkaline Phosphatase (ALP). PQ/Veh/P rats showed lower ALP levels on Days 3 and 6 compared with SA/Veh/P. PQ/Bar/P and PQ/Bar/T rats demonstrated lower levels as early as Day 3 and as late as Day 14. (C) Glucose. SA/Veh/P and PQ/Veh/P rats showed similar glucose levels at all evaluated time points. In contrast, PQ/Bar/P and PQ/Bar/T rats demonstrated lower levels as early as Day 3 and as late as Day 14. (D) Cholesterol. PQ/Veh/P rats showed higher cholesterol levels on Days 3 and 6 compared with SA/Veh/P. PQ/Bar/P and PQ/Bar/T rats demonstrated higher levels at all evaluated time points. (E) Globulins. PQ/Veh/P rats showed higher globulin levels on Day 6 compared with SA/Veh/P. PQ/Bar/P and PQ/Bar/T rats demonstrated higher levels as early as Day 6 and as late as Day 14. (F) Neutrophils. PQ/Veh/P rats showed higher neutrophil levels on Day 6 compared with SA/Veh/P. PQ/Bar/P rats demonstrated higher levels on Day 14. (G) Total CO₂ or bicarbonate. PQ/Veh/P rats showed higher total CO₂ levels on Day 3 compared with SA/Veh/P. PQ/Bar/P rats demonstrated higher levels on Day 3 and lower levels on Day 6. PQ/Bar/T rats demonstrated lower levels on Day 6. (H) Chloride. SA/Veh/P and PQ/Veh/P rats showed similar chloride levels at all evaluated time points. PQ/Bar/P and PQ/Bar/T rats demonstrated higher levels on Day 6. (I) Alanine transaminase (ALT). PQ/Veh/P rats showed lower ALT levels on Day 3 compared with SA/Veh/P. PQ/Bar/P and PQ/Bar/T rats demonstrated higher levels on Days 6 and 14. (J) Total Bilirubin. PQ/Veh/P rats showed higher total bilirubin levels on Day 6 compared with SA/Veh/P. PQ/Bar/P and PQ/Bar/T rats demonstrated higher levels as early as Day 3 and as late as Day 6. ^*p ≤ 0.05 vs. SA/Veh/P or PQ/Veh/T. Error bars are +/− SEM. n = 6–8 rats per study group per time point.

Figure 4

Normalized, endpoint lung weights and histopathology confirmed lung injury and detected effects of bardoxolone treatment. (A) Lung weight to body weight ratios. Compared with SA/Veh/P rats, PQ/Veh/P rats showed higher normalized lung weights at all evaluated time points. ^*p ≤ 0.01 vs. SA/Veh/P. PQ/Bar/P also demonstrated higher normalized weights compared with SA/Veh/P rats on Days 3 and 6. ^*p ≤ 0.0001 vs. SA/Veh/P. (B) Lung histopathology. Lung histopathology confirmed that PQ/Veh/P and PQ/Veh/T rats exhibited alveolar hemorrhage, atelectasis, emphysema, and fibrosis during the course of study. At Day 3, the predominant histological findings were necrosis, loss of type 1 pneumocytes, hemorrhage, and fibrin accumulation within alveoli. Alveolar lumina were multi-focally collapsed and contained abundant hemorrhage, fibrin, and small amounts of necrotic debris (arrows), as well as edema, increased numbers of alveolar macrophages (immune cell infiltrates indicated by stars), and fewer neutrophils. By Day 14, the alveolar septa were expanded by proliferation of cuboidal type II pneumocytes (hyperplasia), fibrin, and varying amounts of fibrous connective tissue (fibrosis indicated by dashed arrows). Furthermore, the alveolar septa were discontinuous with clubbed ends forming large, confluent spaces (emphysema indicated by arrow heads). Perivascular and peribronchiolar connective tissues were expanded by hemorrhage, fibrin, edema, and scattered neutrophils. Rats treated with bardoxolone prophylactically and therapeutically (PQ/Bar/P and PQ/Bar/T, respectively) showed better resolution of alveolar injury. (C–F) Histopathology quantification. (C) Alveolar damage scores. Compared with PQ/Veh/P rats, PQ/Bar/P possessed higher alveolar damage scores at all evaluated time points, while PQ/Bar/T possessed higher scores on Days 6 and 14. (D) Fibrosis scores. Compared with PQ/Veh/P rats, PQ/Bar/P, but not PQ/Bar/T, rats possessed lower fibrosis scores on Day 14. (E) Inflammation scores. Compared with PQ/Veh/P rats, PQ/Bar/T, but not PQ/Bar/P, rats possessed higher inflammation scores on Day 6. (F) Edema scores. All study groups possessed similar edema scores at all evaluated time points. ^*p ≤ 0.05 vs. PQ/Veh/P. Error bars are +/− SEM. n = 6–8 rats per group per time point.

Figure 5

Breathing rate correlated with normalized, endpoint lung weights and histopathology. (A) Lung weight to body weight ratios vs. breathing rate. Breathing rate positively correlated with normalized lung weights for all groups as follows: SA/Veh/P (R = 0.45), PQ/Veh/P (R = 0.71), PQ/Bar/P (R = 67), PQ/Veh/T (R = 0.87), and PQ/Bar/T (R = 0.85); p ≤ 0.05. (B) Alveolar damage score vs. breathing rate. Breathing rate positively correlated with alveolar damage scores, specifically for rats administered PQ and treated with vehicle (PQ/Veh/P; R = 0.76, p ≤ 0.0001). (C) Edema score vs. breathing rate. Breathing rate positively correlated with edema scores, specifically for PQ/Veh/P rats (R = 0.54, p ≤ 0.01). (D) Fibrosis score vs. breathing rate. Breathing rate negatively correlated with fibrosis scores, specifically for rats administered PQ and treated prophylactically with bardoxolone (PQ/Bar/P; R = −0.51, p ≤ 0.05). (E) Inflammation score vs. breathing rate. There was no significant correlation between inflammation scores and breathing rate for all rats administered PQ in which histopathology quantification was performed. n = 22–24 rats per treatment group.