Analgesic Efficacy of Oral Carprofen-Treated Gel and Tablets for an Incisional Pain Model in NSG Mice
Stress in research animals affects their welfare and alters physiologic responses, potentially impacting research outcomes. Laboratory rodents often experience stress from repeated parenteral drug administrations due to handling and needle punctures. In this study, we compared the efficacy of oral carprofen tablets and carprofen-treated nutritional gels compared with injectable carprofen for alleviation of postoperative mechanical and thermal hypersensitivity in NSG mice. Male and female mice (n = 59) were randomly assigned to one of 5 groups: 1) placebo (Placebo tablets or NutraGel, daily); 2) carprofen tablets (CarpTab; 2 mg, daily); and 3) carprofen injection (Carp25SC; 25 mg/kg SC, daily), low-dose carprofen-treated gels (CarpGel-low; 0.11 mg/mL, daily); and high-dose carprofen-treated gels (CarpGel-high; 0.22 mg/mL, daily). Mechanical and thermal hypersensitivities were assessed before surgery on D-1 and at D0 (4 h), D1, and D2 afterward. Plasma carprofen concentration (n = 56) was assessed over 1 to 4 d. Daily clinical observations, fecal occult blood testing, and gross necropsies were performed. Results showed that mechanical and thermal hypersensitivity persisted in the placebo group throughout the study (D0 to D2). CarpTab and Carp25SC groups effectively attenuated mechanical hypersensitivity compared with the baseline (D0 to D2). Compared with baseline, CarpGel-low attenuated mechanical hypersensitivity on D1 and CarpGel-high attenuated mechanical hypersensitivity on D2. All carprofen treatment groups, except CarpGel-high at D0, attenuated mechanical hypersensitivity compared with placebo. Thermal hypersensitivity persisted in all carprofen treatment groups compared with baseline values. However, compared with placebo, CarpTab, Carp25SC, and CarpGel-low provided attenuation of thermal hypersensitivity. Plasma carprofen concentrations did not significantly differ among oral formulations. Fecal occult blood testing was positive only in 1 of 6 mice in the CarpGel-high group at 1 and 2 d after administration. These findings support the use of orally administered CarpTab as an alternative analgesic to Carp25SC for incisional pain in NSG mice.
Introduction
It is widely recognized that the stress experienced by research animals not only impacts their welfare but also alters their physiologic responses, potentially affecting research outcomes.8,9,16 One common source of stress for research animals is the repetitive process of parenteral drug administration. This stress is primarily induced by the handling required for the injections and the discomfort of needle punctures. The majority of analgesic medications administered to laboratory mice are given parenterally, with some requiring one to 3 daily doses to ensure adequate pain relief throughout the day. This repetitive handling and injection process can significantly impact both animal welfare and experimental outcomes.
To address this concern, stress-free, self-administrable, oral dosing methods have been explored and used effectively in rodents.18,31,40,55,67,68 Medicated supplemental diets, such as flavored nutritious tablets87 or flavored diet gels,19,67 and the incorporation of medications into drinking water18,31,34,40,42 provide alternatives that minimize handling-related stress for both the researchers and the animals. While rodents may display preferences for specific medications in their drinking water,31,40 the palatability of flavored nutritional supplements encourages consumption, stimulates appetite, and prevents complications such as postoperative body weight loss29 arising from a decreased interest in standard animal diets while serving the intended medicinal purpose.
Nonsteroidal anti-inflammatory drugs (NSAIDs), such as carprofen and meloxicam, are frequently used analgesic medications to alleviate mild to moderate pain associated with inflammation in rodents.24,49,60,83 These medications are often administered either independently or in combination with opioids (for example, buprenorphine) or local anesthetics (for example, lidocaine or bupivacaine) to offer multimodal analgesia.1,60,61,84 Carprofen is commonly administered parenterally in rodents; however, it has recently become available in flavored nutritional tablet form12 and can also be added to drinking water.18,31,40,61 Carprofen works by inhibition of cyclooxygenase (COX) enzymes, specifically COX-1 and COX-2.24,49,78 Blocking these enzymes prevents the synthesis of prostaglandins, which are a key mediator of pain and inflammation.24,49,78 While this mechanism allows carprofen to alleviate pain, overdose or prolonged exposure to carprofen can lead to gastrointestinal ulceration, renal toxicity, hepatic damage, and impaired platelet function.3,49,51,69,75,78 Because of the potential adverse effects, it is critical that the dose administered is effective while also not being too high to cause adverse effects.
Finding effective analgesic medication is a notable challenge, particularly for the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse strain. NSG mice are a widely used strain in laboratory research due to their unique immunodeficient characteristics. These mice are severely immunocompromised, lacking functional T cells, B cells, and natural killer cells,73,74 and they are used to study various aspects of human biology, including cancer, infectious diseases, and autoimmune disorders.13,72–74 A recent study involving NSG mice revealed that commonly used opioid analgesics were ineffective in reducing both mechanical and thermal hypersensitivity in a plantar incisional model.6 However, our recent investigation demonstrated that carprofen, administered at a dose of 25 mg/kg SC once a day, successfully alleviated both types of hypersensitivity for up to 2 d using the same model and experimental conditions.3 In this current study, we aimed to assess the efficacy of oral carprofen by using self-administration of flavored carprofen tablets and carprofen-treated diet gels as a means to reduce the stress associated with parenteral administration in NSG mice. To our knowledge, the effectiveness of self-administered oral carprofen has not been assessed previously in mice or in, more specifically, immunodeficient mouse strains.
The aims of this study were to 1) evaluate the efficacy of self-administered oral carprofen using medicated tablets, and low-dose (0.11 mg/mL) and high-dose (0.22 mg/mL) carprofen-treated gels in NSG mice over a 48-h period; 2) determine plasma concentrations of these self-administered oral carprofen doses at various time points; and 3) evaluate for evidence of carprofen toxicity using fecal occult blood tests and gross necropsies. We hypothesized that both a low and high dose of carprofen-medicated gels and carprofen tablets would offer similar analgesia as the injectable form of carprofen at a 25-mg/kg dose. Furthermore, we hypothesized that the low-dose carprofen-treated gel and the carprofen tablets would induce lower levels of toxicity than the high-dose carprofen-treated gels.
Materials and Methods
Mice.
Adult (mean age: 12 wk [range: 8 to 30 wk]) male (mean weight: 32.4 g, [range: 25 to 37 g]) and female (mean weight: 31.1 g, [range: 22 to 43 g]) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were used to test hypersensitivity (n = 59), measure plasma carprofen concentrations (n = 56), and evaluate fecal occult blood (n = 30). Mice used in this study were sourced from a closed colony of NSG/J mice, bred in-house in a barrier facility. The breeding stock for this colony originated from The Jackson Laboratory (Bar Harbor, ME). Breeder mice are replaced every 8 months, and genotyping is not routinely performed. Before the study, mice were group housed by sex in the barrier facility with disposable, irradiated IVC cages (Innovive, San Diego, CA) prefilled with Alpha-Dri bedding (Shepherd Specialty Papers, Kalamazoo, MI) enriched with Enviro-dri (Lab Supply, Fort Worth, TX) and paper tubes (Pacific Paper Tube, Stockton, CA). The mice colony was surveilled by dirty bedding sentinels every 4 months for various pathogens and parasites and were free of lymphocytic choriomeningitis virus, mouse hepatitis virus, mouse parvovirus, Sendai virus, ectromelia virus, mouse rotavirus, minute virus of mice, pneumonia virus of mice, Theiler murine encephalomyelitis virus, murine adenovirus 1 and 2, Helicobacter spp., reovirus, murine norovirus, Rodentibacter pneumotropicus (Pasteurella pneumotropica), Mycoplasma pulmonis, pinworms, and endo- and ectoparasites.
To maintain consistent testing and housing environmental conditions, approximately one week before the experiment, mice were relocated and acclimated to a holding room equipped for daily hypersensitivity testing. The room adhered to a 12:12-h dark:light cycle, with temperatures ranging from 68 to 79 °F (20 to 26 °C) and relative humidity between 30% and 70%. Mice had ad libitum access to autoclaved commercial rodent diet (Teklad Global; 18% Protein Rodent Diet 2018SX; Inotiv, Indianapolis, IN) and chlorinated water (Aquavive; Innovive). Animal cages were changed once a week on a regular basis. All experimental procedures were conducted with approval from the Stanford University IACUC (Administrative Panel for Laboratory Animal Care), adhering to the Guide for the Care and Use of Laboratory Animals.41
Medicated diet gel.
Different thermoreversible gels were given to a separate group of NSG mice to check the more preferred and palatable gel (n = 8). NutraGel (Figure 1A; no. NGB-2; Bio-Serv, Flemington, NJ) was found to be more palatable and therefore was used for the study. NutraGel is a nutritionally complete, bacon-flavored diet gel that comes in 2-oz cups.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-118
In this study, we administered oral carprofen in 2 dosage groups: the CarpGel-low group targeting a 25-mg/kg/day dose and the CarpGel-high group targeting a 50-mg/kg/day dose. Each 2-ounce gel cup contained approximately 60 mL of gel. Assuming an average daily gel consumption of 7 mL per 30 g mouse,21 6.45 mg of carprofen was added to each 60 mL gel cup for the CarpGel-low group, and 12.9 mg was added for the CarpGel-high group. To achieve these concentrations, 0.13 mL and 0.26 mL of carprofen from a 50-mg/mL stock solution (Norbrook Laboratories; Rossmore Industrial Estate, Ireland) were added to the 2-ounce gel cups for the CarpGel-low and CarpGel-high groups, respectively. The final carprofen concentrations in the gel were 0.11 mg/mL for the CarpGel-low group and 0.22 mg/mL for the CarpGel-high group. The gel diet was prepared one week before starting the experiment as described previously for carprofen-containing sucralose gel.21 Briefly, the gel was liquified by preheating it in a water bath at 55 °C for 20 min. Once liquified, the top surface of the lid was aseptically cleaned using alcohol wipes. Then, 0.13 mL or 0.26 mL of carprofen dosage was taken from the carprofen-injectable stock solution and was injected into the gel cups by piercing the lid with 25-G needle on a 1-mL syringe. The injection hole on the lid was closed with tape and the gel-drug mix was shaken vigorously for a minimum of 60 s to ensure even distribution of the drug in the gel. The medicated gels were then placed in a refrigerator for 1 h until solidification.
Mice were singly housed for ease of tracking their consumption and were acclimated to the gel diet for 2 d before the surgery. Fifteen grams of placebo or medicated NutraGel gel was placed daily on 7 ml small weigh boats (ClearH2O, Westbrook, ME) (Figure 2A). Gels were cut into small pieces using a wooden applicator stick and placed in cages with a cup holder (ClearH2O). The gel diet was replaced daily, and the weights of the uneaten gels were recorded. Mice that did not eat at least 3 g of gel were removed from the study (n = 2 hypersensitivity evaluation; n = 1 plasma evaluation).
Medicated flavored tablet.
Bacon-flavored 2-mg carprofen tablets (Figure 1B; Rimadyl; no. MD150-2; Bio-Serv) were used for this study. Mice were singly housed as in the gel consumption experiment. Carprofen and placebo tablets were placed on weigh boats and then kept in cages starting 2 d before the surgery (Figure 2B). Mice were excluded from the study if there was no consumption of the tablet before the surgery, on D-1 (n = 9 for hypersensitivity testing, n = 4 for plasma analysis).
General experimental design.
This study consisted of 3 experiments: 1) hypersensitivity testing after plantar incisional surgery, 2) carprofen plasma concentration determination, and 3) assessment of gastric ulceration using fecal occult blood testing and gross necropsy. All the treatments in this study were provided continuously throughout the study period in addition to the ad libitum chow pellets and purified water.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-118
Experiment 1. Hypersensitivity testing.
Study design.
The experimental timeline for hypersensitivity assessment is shown in Figure 3A. Male (n = 44) and female (n = 15) NSG mice were used for this study. Two days before surgery (D-2), mice were singly housed and provided with extra enrichment such as shredded paper, Enviro-dri (Lab Supply) and paper tubes (Pacific Paper Tube) to minimize stress. Mice were randomly assigned into 5 groups using random number generators and acclimated to the gels or the tablets for the 2 d before surgery. The groups were 1) placebo gel (no. NGB-2; Bio-Serv) or placebo tablet (no. F05266; Bio-Serv) (Placebo; n = 12 [9 males, 3 females]); 2) subcutaneous injection of carprofen at the 25-mg/kg dose (Carp25SC; n = 12 [9 males, 3 females], once daily for 2 d; Norbrook Laboratories); 3) carprofen tablets (CarpTab; 2 mg timadyl, n = 11 [8 males, 3 females]; no. MD150-2; Bio-Serv); 4) low dose of carprofen gel (0.11 mg/mL; CarpGel-low; n = 12 [9 males, 3 females]; no. NGB-2; Bio-Serv); and 5) high dose of carprofen gel (0.22 mg/mL; CarpGel-high, n = 12 [9 males, 3 females]; Bio-Serv). One mouse was excluded from the study prior to the start of the experiment due to a health issue unrelated to the study, resulting in the CarpTab group having 11 mice. Mice in group 1, served as controls, and were given either placebo tablets or unmedicated gels ensuring representation of both types of oral treatments. Each day, the uneaten gels and tablets were weighed and recorded at approximately 0900 and replaced with new previously prepared gel or tablets to avoid contamination of the treatments with bedding material and feces. Animal weights were recorded daily, allowing calculation of doses of carprofen (mg/kg) received by the individual animal each day during the experiment. Animals in group 3 that received injectable carprofen also received either placebo gels or tablets to keep the experimenter blind to the treatment group. Mice in this group received subcutaneous injections of carprofen 5 to 10 min before the surgery and at 23- and 47-h time points, an hour before the hypersensitivity testing with 1 mL tuberculin syringes with a 25-G needle. During drug administration, mice were manually restrained by scruffing the loose skin around the back of the neck and between shoulders and by holding the tail for stabilizing the mice for the injection. During routine husbandry care, mice in this study were briefly handled by the tail for tasks such as cage changing and separation. During testing, mice were handled either by the tail or by means of a tunnel handling method using the paper tubes provided for enrichment purposes.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-118
Baseline mechanical and thermal hypersensitivity data were collected and recorded between 0900 and 1000, one day before surgery (D-1). On the day of surgery (D0), following paw incisional surgeries performed in the morning between 0900 and 1000, hypersensitivity tests were conducted at 4 h (D0), 24 h (D1), and 48 h (D2) postsurgery by the same experimenter who was blinded to the treatments. On D2, after the completion of the last hypersensitivity tests, mice were euthanized with carbon dioxide asphyxiation, and a necropsy was performed to assess gross pathology.
Mechanical hypersensitivity.
Mechanical hypersensitivity was assessed using the Von Frey monofilament nociceptive assay, following procedures described previously.3,4,6,59,87 Briefly, mice were acclimated with the testing environment and in clear plastic chambers (10.1 × 10.1 × 12.5 cm) placed on an elevated wire mesh platform (Electronic von Frey Mesh Stand; IITC Life Science, Woodland Hills, CA). A calibrated Semmes-Weinstein von Frey filament (0.4 g) was applied to the contralateral (control) and ipsilateral (test) hind paws, exerting mechanical stimulus 10 times each. The filament made brief contact (1 to 2 s) with random areas of the paws, excluding the heel, pads, and toes. The test was performed with an interstimulus interval of approximately 5 min. Responses such as withdrawal, shaking, or licking of the stimulated paw were noted as positive, while the absence of such responses was recorded as negative. Mechanical hypersensitivity was defined as a significant increase in the frequency of paw withdrawals following a mechanical stimulus as compared with baseline.
Thermal hypersensitivity testing.
Responses to thermal stimuli were assessed using the Hargreaves nociceptive assay, in accordance with previously established procedures.3,4,6,59,87 The test was always performed following the completion of mechanical assessments. Briefly, mice were placed in clear plastic chambers (10.1 × 10.1 × 12.5 cm) atop a preheated glass (29 °C) platform after the mechanical tests, allowing a 15-min acclimation period. A focused thermal stimulus, generated by a 50-W light bulb (Plantar Analgesia Meter; IITC Life Science) was applied to the plantar surface of each hind paw with a 25% beam intensity. Each paw underwent 4 trials, separated by at least 5 min, with the contralateral paw serving as a control. To prevent tissue injury, a maximum cutoff time of 20 seconds was implemented. The same criteria were used as the mechanical hypersensitivity assay to determine positive and negative responses. Mean withdrawal latency was calculated based on the average of the last 3 trials, excluding the first trial in all instances. Thermal hypersensitivity was defined as a significant decrease in paw withdrawal latency caused by the onset of focal thermal stimuli as compared with baseline.
Surgery.
Surgical procedures were conducted as described previously.3,6,14,59,64,77 Briefly, mice were anesthetized using 2% to 2.5% isoflurane. Subcutaneous administration of a single dose of Cefazolin (30 mg/kg SC; GlaxoSmithKline, Research Triangle Park, NC) and prewarmed 0.9% saline (5 mL/kg SC) was performed. The left paw’s plantar surface was meticulously prepared in an aseptic manner. A 0.5-cm skin incision was made along the plantar aspect of the paw and a curved iris tissue forceps was used to elevate the plantaris muscle. The muscle was longitudinally incised without disrupting its attachment, and a second curved iris tissue forcep was introduced into the incision. A gentle lateral grip was applied for 10 s and both forceps were withdrawn. A drop of sterile saline was applied, and excess fluid was removed using sterile cotton-tipped swabs. The skin incision was closed using 4-0 silk suture. Anesthesia depth was monitored using the paw withdrawal reflex, along with assessments of mucous membrane color and respiration throughout the surgical period. Mice were allowed to recover in a warm environment before being returned to their home cage until the hypersensitivity tests.
Clinical observations and gross pathology.
Any abnormal behaviors or clinical signs such as altered activity levels, mobility, limping, dehydration, paw licking, scratching, or biting were evaluated twice daily (during hypersensitivity testing and in the afternoon between 1500 to 1600). At the completion of the study, mice were euthanized, and gross necropsy was performed.
Experiment 2: Plasma drug concentration analysis.
Study design.
A separate group of mice were housed, grouped, and treated as described above in experiment 1. Plasma samples from male (n = 27) and female (n = 29) mice were collected via a retroorbital technique to determine carprofen plasma concentration. Two days before blood collection (D-2), male and female mice were randomly grouped into one of 4 groups using random number generators to receive placebo or medicated gels or tablets. Both sexes were represented at each plasma collection time point, and none of the mice in this experiment underwent plantar surgery. The groups were 1) Placebo; n = 2 (1 male, 1 female); 2) CarpTab; n = 18 (9 males, 9 females); 3) CarpGel-low; n = 18 (12 males, 6 females); and 4) CarpGel-high; n = 18 (12 males, 6 females).
During plasma collection, terminal blood collection was performed using a retro-orbital technique. Briefly, mice were anesthetized in an induction chamber using 4% to 5% isoflurane in 100% oxygen. Upon achieving a surgical plane of anesthesia, confirmed by a toe pinch reflex test, 0.7 to 0.8 mL of whole blood was collected with a nonheparinized capillary tube. Euthanasia was conducted via exsanguination, followed by cervical dislocation as a secondary method. Blood samples were centrifuged at 3,451 × g for 20 min in a microcentrifuge. The resulting plasma was carefully extracted, transferred to 1-mL cryogenic tubes, and stored at −80 °C until further analysis. To determine if animals receiving 2 d of medicated gel/tablet achieve comparable plasma concentrations with animals receiving injectable carprofen, blood collection was carried out at 2, 4, 12, and 23 h on D0 after 2 d of oral carprofen exposure. In addition, to determine plasma concentration after extended exposure to the gels/tablets, a blood sample was collected at 24 h (D1) and 48 h (D2) after 3 and 4 d of oral carprofen exposure, respectively. Three mice per group per time point underwent terminal blood collection except for the placebo group where a blood sample was collected from 2 mice at the 2 h time point. Plasma collection, processing, and carprofen concentration analysis were conducted as previously described.3 The experimental timeline is shown in Figure 3B.
Plasma carprofen concentration analysis.
Plasma carprofen concentration was measured by the Pharmaceutical Sciences Research Institute at the McWhorter School of Pharmacy (Samford University, Birmingham, AL) using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).
Approximately 0.25 mL of plasma was collected from each mouse and shipped overnight on dry ice. Calibration standards, blanks, and quality controls were prepared by spiking 20 μL of mouse plasma with the appropriate amount of carprofen to achieve plasma concentrations that ranged from 1 to 500 μg/mL. Standards, blanks, quality controls, and samples were spiked with 10 μL of 100 μg/mL ketoprofen in acetonitrile as an internal standard. To precipitate proteins, 200 μL of acetonitrile containing 0.1% formic acid was added to samples. The samples were mixed by vortexing and centrifuged for 5 min at 21,130 × g. The supernatant was then transferred to 0.5-mL autosampler vials and analyzed by HPLC with UV detection at 254 nm. Chromatographic separation of the compounds was achieved using a Shimadzu 2010 HPLC system (Shimadzu Scientific Instruments, Columbia, MD) with a 150 × 4.6 mm Luna C18 reverse phase column (Phenomenex, Torrance, CA) at ambient temperature. Mobile phase A consisted of deionized water containing 0.1% formic acid, and mobile phase B consisted of Acetonitrile. The carprofen and the internal standard (ketoprofen) were analyzed using a gradient elution profile in which mobile phase B was held at 50% for 1 min, then increased to 90% over 4 min, held at 90% for 1 min, returned to 55% over 0.5 min, and equilibrated for 3.5 min. The flow rate was set at 1 mL/min and 10 μL was injected onto the column. Carprofen was eluted at 5.4 min and ketoprofen at 4.4 min. To reliably determine carprofen concentrations, the lower limit of detection was set at 0.6 µg/mL.
Experiment 3.
Toxicity assessment: Fecal occult blood test.
A distinct cohort of mice (n = 30) underwent the housing and treatment regimens as those in experiments 1 and 2 to evaluate for fecal occult blood. The groups were 1) Placebo; (n = 6, 3 males and 3 females); 2) CarpTab (n = 6, 3 males and 3 females); 3) Carp25SC, (n = 6, 3 males and 3 females); 4) CarpGel-low, (n = 6, 3 males and 3 females); and 5) CarpGel-high (n = 6, 3 males and 3 females). Fecal occult blood tests were conducted promptly following fecal sample collection at various time points. Baseline data were collected on D-2 immediately before the gels or tablets were provided and on D0 immediately before carprofen administration for Carp25SC group. The tests were performed again after 2 d of gels/tablets were provided on D0, 24 h (D1), and 48 h (D2). For Carp25SC group, the tests were performed at 4 h after drug administration (D0) and at 24 (D1) and 48 h (D2) one hour after the second and third doses were administered at 23 and 47 h, respectively. Fecal samples were collected and processed as described previously.3 Fecal occult blood testing was performed using the Hemoccult test kit (Beckman Coulter, Bree, CA) according to manufacturer’s instructions. The experimental timeline is shown in Figure 3C.
Statistical analysis.
Statistical analysis was conducted by combining data from male and female mice due to an unequal distribution of animals across the treatment groups. Analysis was performed using 2-way repeated measures ANOVA with Bonferroni correction for multiple comparisons66 to detect significant differences within and between groups and over time. Data are expressed as means ± SEM. A P value of less than 0.05 was considered significant.
Group size determinations.
This study was conducted on 5 treatment groups with 4 repeated time points. Before the experiment, the sample size was calculated (0.20 effect size, 0.25 correlation among repeated measurements, and 0.05 level of significance) and resulted in at least 11 mice per treatment with approximately 82% power of the test. The assumption about multivariate normality of residuals was also investigated and residuals for all treatment groups followed multivariate normal distributions.
Results
Body weight.
There were no significant changes in body weight among treatment groups or when compared with the respective baseline values of each group at any time point during the study.
Experiment 1: Hypersensitivity testing.
Responses to mechanical hypersensitivity tests.
A day before the surgery, baseline (D-1) values of ipsilateral (surgical) and contralateral (control) hind paw responses were not significantly different between groups. Following surgery, the placebo group’s ipsilateral hind paws showed a significantly higher mechanical hypersensitivity response than the baseline (D-1) value of the same group throughout the study (D0, D1, and D2; P < 0.0001) (Figure 4; Table 1). In the CarpGel-low and CarpGel-high groups, mechanical hypersensitivity was significantly higher on D0 (P = 0.0058) and D2 (P = 0.0154) and on D0 (P = 0.0030) and D1 (P = 0.0333), respectively, compared with their respective baseline values. However, in the CarpTab and Carp25SC groups, mechanical hypersensitivity was attenuated throughout the study (D0, D1, and D2) and no significant differences were present when compared with their baseline values. Mechanical hypersensitivity in the placebo group was significantly higher than that of the CarpTab (D0: P = 0.0112; D1: P = 0.0001; D2: P = 0.0002), Carp25SC (D0: P = 0.0067; D1 and D2: P < 0.0001), CarpGel-low (D0: P = 0.0373; D1: P < 0.0001; D2: P = 0.0022), and CarpGel-high groups, except at D0 (D1: P = 0.0003; D2: P = 0.0004). There were no significant differences in mechanical hypersensitivity attenuation between the carprofen groups at any time point during the study.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-118
| Time points | Placebo (n = 12) | CarpTab (n = 11) | Carp25SC (n = 12) | CarpGel-low (n = 12) | CarpGel-high (n = 12) |
|---|---|---|---|---|---|
| Mechanical hypersensitivity (no. of withdrawals of ipsilateral hind paw; mean ± SEM) | |||||
| D-1 | 2 ± 0.17 | 1.91 ± 0.25 | 1.75 ± 0.22 | 1.5 ± 0.26 | 1.67 ± 0.19 |
| D0 | 6.42 ± 0.6 | 3.73 ± 0.68 | 3.67 ± 0.58 | 4.08 ± 0.73 | 4.42 ± 0.68 |
| D1 | 7.42 ± 0.65 | 3.64 ± 0.56 | 3.67 ± 0.62 | 3.58 ± 0.91 | 4 ± 0.62 |
| D2 | 7 ± 0.65 | 3.45 ± 0.51 | 3.08 ± 0.51 | 4 ± 1.03 | 3.67 ± 0.57 |
| Thermal hypersensitivity (thermal latency of ipsilateral hind paw measured in seconds; mean ± SEM) | |||||
| D-1 | 18.12 ± 0.55 | 18.13 ± 0.85 | 17.12 ± 0.86 | 15.51 ± 0.84 | 16.34 ± 0.76 |
| D0 | 3.99 ± 1 | 8.38 ± 1.49 | 10.77 ± 1.65 | 7.58 ± 1.26 | 6.05 ± 1.29 |
| D1 | 2.91 ± 0.31 | 10.96 ± 1.91 | 9.63 ± 1.64 | 7.44 ± 1 | 5.68 ± 1.03 |
| D2 | 2.97 ± 0.33 | 7.64 ± 0.99 | 11.91 ± 1.85 | 7.97 ± 1.27 | 6.17 ± 1.07 |
Contralateral mechanical hypersensitivity (Figure 5) was not significantly different from the baseline (D-1) value for the placebo, CarpTab, CarpGel-low, or CarpGel-high at any time point throughout the study. Contralateral hypersensitivity was significantly increased in the Carp25SC group only on D2 (P = 0.01016) as compared with the baseline value (D-1). Mechanical hypersensitivity did not differ between treatment groups at any other time points.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-118
Responses to thermal hypersensitivity tests.
Baseline (D-1) values of both ipsilateral and contralateral hind paw responses were not significantly different between groups.
Following surgery, the placebo group ipsilateral hind paws showed a significantly lower thermal latency than the baseline (D-1) value of the same group throughout the study (D0, D1, and D2; P < 0.0001) (Figure 6; Table 1). Similarly, thermal latency significantly decreased in all the carprofen groups throughout the study when compared with their respective baseline values (CarpTab: D0 and D2: P < 0.0001; D1: P = 0.0028; Carp25SC: D0: P = 0.0031; D1: P = 0.0009; D2 and D0: P = 0.0121; CarpGel-low: D0 and D2: P = 0.0001; D1: P = 0.0003; CarpGel-high: D0, D1, and D2: P < 0.0001). However, compared with the placebo group, thermal latency was significantly higher in CarpTab group (D1: P < 0.0001; D2: P = 0.0496), Carp25SC group (D0: P = 0.0004; D1: P = 0.0005; D2: P < 0.0001), and CarpGel-low group (D2: P = 0.0213). In the CarpGel-high group, thermal latency was not significantly different from the placebo group at any time point during the study. There were also significant differences in thermal hypersensitivity attenuation between carprofen-treated groups. The thermal latency was significantly different between the Carp25SC and CarpGel-high groups on D0 (P = 0.0368) and D2 (P = 0.0045), and between the and CarpGel-high groups on D1 (P = 0.0151).
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-118
In the contralateral hind paws (Figure 7), only the CarpGel-low group showed a significant difference in thermal latency from its respective baseline value on D1 (P = 0.0219). The thermal latency measured in the carprofen groups did not differ from the placebo group at any time point. However, when comparing between carprofen groups, thermal latency was significantly different between the Carp25SC and CarpGel-high groups on D2 (P = 0.0498).
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-118
Tablet and gel consumption.
The percentage of tablet or gel consumption did not differ significantly between male and female mice at any time point when compared within the same groups. The average consumption of carprofen-treated tablets and gels from D-1 to D2 was as follows: CarpTab: 58.26%; CarpGel-low: 48.82%; and CarpGel-high: 31.65%. Tablet or gel consumption did not significantly differ between carprofen treatment groups. In addition, the rate of consumption was not significantly different before (D-1 to D0) and after surgery (D1 to D2).
Dose of carprofen consumed.
The average dose of carprofen ingestion is summarized in Figure 8 and Table 2. The average dose of carprofen ingested from D-1 to D2 in the CarpTab, CarpGel-low, and CarpGel-high groups was 36.78, 27.03, and 49.08 mg/kg, respectively. The dose of carprofen ingested was not significantly different before and after surgery. However, the dose of carprofen differed significantly between the different treatment groups. In the CarpTab group, the dose of carprofen (33.4 mg/kg) was significantly different from the CarpGel-high group (53.6 mg/kg; P = 0.0305) on D-1. The dose of carprofen received by mice in CarpGel-low and CarpGel-high groups was significantly different (P = 0.0143) throughout the study (D-1 to D2). Compared with the injectable carprofen group (Carp25SC), mice in the CarpGel-high group received a significantly higher dose of carprofen throughout the study (D0, D1, and D2; D-1 values were not analyzed since the Carp25SC group dosing started on D0).
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-118
| Dose of carprofen consumed (mg/kg) | |||||
|---|---|---|---|---|---|
| Dose | D-1 | D0 | D1 | D2 | Average |
| CarpTab (n = 11) | 33.44 | 41.74 | 35.41 | 36.54 | 36.78 |
| CarpGel-low (n = 12) | 33.58 | 29.31 | 20.21 | 25.03 | 27.03 |
| CarpGel-high (n = 12) | 53.59 | 51.51 | 42.21 | 49.03 | 49.08 |
Clinical observation and gross pathology.
No abnormal behaviors were observed except for licking of the incised (left hind) paw (8 of 12 mice in the Placebo, 5 of 11 mice in CarpTab, 7 of 12 mice in Carp25SC, 2 of 12 mice in CarpGel-low, and 4 of 12 mice in CarpGel-high groups) and/or limping (8 of 12 mice in the Placebo, 1 of 11 mice in CarpTab, 2 of 12 mice in Carp25SC, 4 of 12 mice in CarpGel-low, and 4 of 12 mice in CarpGel-high groups) during the study period. At the completion of hypersensitivity tests, mice were euthanized via carbon dioxide inhalation, and a gross necropsy was performed. No abnormal gross findings were observed in any of the mice.
Experiment 2: Plasma drug concentration analysis.
Plasma samples were collected at various time points from a separate group of mice that consumed carprofen-treated gels and tablets. Two placebo-treated animals were used as a negative control (data not shown). Mice received carprofen-treated gels and tablets for 2 d before the start of any blood collection and continued for 2 d afterward, depending on their scheduled time point for blood collection. Blood samples were collected at 2, 4, 12, and 23 h after 2 d of oral carprofen exposure on D0 and 24 (D1) and 48 (D2) h after 3 and 4 d of carprofen exposure, respectively.
Carprofen plasma concentrations were variable (Figure 9; Table 3), with some samples showing no peak concentrations (values less than 0.6 μg/mL) (CarpTab: 1 mouse at 12 h; CarpGel-low: 2 mice at 2 and 24 h; and CarpGel-high: 2 mice at 23 h, 1 mouse at 2 h, and 1 mouse at 24 h) and others displaying as high as 126 μg/mL. Average plasma concentrations did not differ between groups at any time point. When comparing time points within each carprofen group, only CarpTab’s plasma concentration was significantly different between the 12 and 24 h time points (P = 0.0196).
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-118
| Plasma carprofen concentration (μg/mL; mean ± SEM) | |||
|---|---|---|---|
| Time points (h) | CarpTab (n = 3 per time point) | CarpGel-low (n = 3 per time point) | CarpGel-high (n = 3 per time point) |
| 2 | 55.6 ± 26.68 | 19.15 ± 3.55 | 49.3 ± 25.94 |
| 4 | 36.13 ± 4.83 | 8.4 ± 6.56 | 69.85 ± 53.15 |
| 12 | 33.6 ± 28.7 | 27.93 ± 5.54 | 48.83 ± 17.4 |
| 23 | 37.43 ± 7.35 | 55.2 ± 36.89 | 81.3 ± 0 |
| 24 | 80.87 ± 17.68 | 46.5 ± 24 | 72.8 ± 30.1 |
| 48 | 37.63 ± 20.51 | 28.1 ± 19.62 | 21.73 ± 4.04 |
Experiment 3: Fecal occult blood test.
Fecal occult blood tests were performed on a separate group of mice that received the same treatments as in experiments 1 and 2 to determine the presence of occult blood in their feces. Tests were conducted before the administration of any treatments and on D0, D1, and D2 after drug administration. Fecal occult blood was detected only in 1 of 6 mice receiving with CarpGel-high on D1 and D2 but not in any other carprofen-treated mice.
Discussion
To our knowledge, this is the first study that evaluates the efficacy of self-administered oral carprofen in NSG mice. The aims of this study were to 1) evaluate the efficacy of self-administered oral carprofen via medicated tablets or low-dose (0.11 mg/mL) and high-dose (0.22 mg/mL) carprofen-treated gels over 48 h; 2) determine plasma concentrations of these self-administered oral carprofen doses at various time points; and 3) assess carprofen toxicity using fecal occult blood tests and gross necropsies.
In the present study, after 4 d of continuous exposure to the medicated tablets or carprofen-treated gels, we demonstrated that CarpGel-low and CarpGel-high groups attenuated mechanical hypersensitivity on D1and D2, respectively, while CarpTab and Carp25SC groups provided consistent attenuation throughout the study compared with their presurgery baseline values. All carprofen groups, except the CarpGel-high group on D0, showed significant mechanical hypersensitivity reduction compared with the placebo group. Thermal hypersensitivity was significantly reduced in the CarpTab group (on D1 and D2), the Carp25SC group (on D0, D1, and D2), and the CarpGel-low group (on D2) when compared with the placebo group. Carprofen plasma concentrations were variable, and fecal occult blood was detected only in 1 of the 6 mice treated with CarpGel-high on D1 and D2.
Rodent handling, particularly during drug administration, is a significant source of animal stress, primarily due to the need for restraint techniques such as tail handling and scruffing to immobilize the animal.10,39 Tail handling in mice is known to induce stress responses, including increased anxiety, heightened corticosterone levels, and aversion to human interaction, which can interfere with experimental outcomes and animal welfare.25,32,33,71 It is a common practice to physically restrain animal subjects as a stress-inducing mechanism in stress-related research.7,35,62,63 Therefore, the stress caused by restraint should be regarded with importance in biomedical research, as it can cause reliable and measurable changes in the physiology of the animal.10,15,35 For example, in a study using female, hairless SKH-1 mice, restraint stress has been reported to delay cutaneous wound healing.62 In addition, during drug administration repeated needle punctures can further increase handling stress, particularly in animals that have already undergone stressful procedures such as surgery. Stress alters the physiology of the animal by activating the hypothalamic-pituitary (HPA) axis and the sympathetic nervous system.26,37,76,79 Once the brain interprets stress and HPA is activated, corticotropin-releasing hormone is released from the hypothalamus, which in turn activates the pituitary gland to release ACTH.37,76,79 The ACTH then triggers adrenal glands to release the stress hormone, cortisol, which causes several physiologic changes including changes in cardiovascular, respiratory, gastrointestinal, renal, and endocrine systems.7,10,15,37,39,53,54,76,79,85
Carprofen is an NSAID available in different formulations such as injectable, or orally in flavored chewable tablets, mixed into gel diets, or added to drinking water.3,18,27,40,52,61 Our recent work with injectable carprofen solution revealed that subcutaneously administered carprofen at a 25-mg/kg dose effectively attenuated mechanical and thermal hypersensitivity for at least 2 d in NSG mice using a paw incisional pain model.3 This finding was particularly significant as it demonstrated the efficacy of carprofen, in contrast to the commonly used buprenorphine formulations that failed to attenuate both mechanical and thermal hypersensitivity for NSG mice.6
One concern when implementing orally administered drugs would be the first-pass effect.38 The first-pass effect or presystemic metabolism is a pharmacological phenomenon where the drug first passes through the gastrointestinal tract (GIT) after ingestion and is then absorbed and carried into the liver to be metabolized. Metabolism in the liver causes extensive reduction in the bioavailability and thus contributes to subtherapeutic action.38 Carprofen is a member of aryl propionic acids, and it is able to be rapidly and nearly completely absorbed into systemic blood circulation after oral administration with minimum effect of first-pass metabolism in the bioavailability.46 Due to its moderate to high lipid solubility49 and reduced impact of first-pass metabolism,46 carprofen has high bioavailability after oral dose administration in several species of animals (see examples: 78.66% in rainbow trout,80 horses 75% to 100%,22 and sheep 62.51%23). Because of the high oral bioavailability, we expected that carprofen doses similar to what was previously found to be efficacious for NSG mice3 would be again effective in an oral formulation. However, the results of this study indicate that additional research is necessary to establish clinically effective carprofen doses for gel preparations to achieve optimal plasma concentrations and therapeutic effects while minimizing toxicity in NSG mice.
In the current study, we compared the efficacy of orally administered bacon-flavored carprofen medicated chewable tablets (CarpTab) and carprofen-treated nutritional gels (CarpGel) with injectable carprofen at a 25-mg/kg dose (Carp25SC) in NSG mice. Our finding indicated that CarpTab and Carp25SC provided mechanical hypersensitivity attenuation over 2 d postsurgery consistent with the injectable carprofen at a 25-mg/kg dose in our previous study with NSG mice.3 This is consistent with another study using C57BL/6 N and CD-1 mice, where carprofen administered both subcutaneously and orally (via drinking water) at doses of 10 and 25 mg/kg, effectively reduced mouse grimace scale pain scores within the first 24 h after surgery.18 Similarly, subcutaneously administered carprofen at the 20- and 25-mg/kg doses reduced the mean score of a mouse grimace scale in CD-1 mice after laparotomy.56 In the present study, the estimated average dose of carprofen ingested by CarpTab-treated mice was 36.78 mg/kg, which is slightly higher than the 25-mg/kg dose in the Carp25SC group. The plasma concentration of carprofen provided by the tablets may not be as high as the concentration that could be induced by a parenteral 36.78-mg/kg dose, due to differences in oral bioavailability. Our results indicate that the oral-administered carprofen tablet (1.17 mg) is sufficient to attenuate mechanical hypersensitivity for a minimum of 2 d in adult NSG mice. These studies support the use of orally administered carprofen at the dose used to attenuate induced and spontaneous pain in mice.
The findings were not as consistent in the CarpGel-low and CarpGel-high groups. The lack of efficacy in the CarpGel-low group could be due to the lower oral carprofen dose (27.03 mg/kg) compared with the CarpTab group (36.78 mg/kg). On the contrary, the lack of efficacy in the CarpGel-high group might be attributed to the development of toxicity, as the estimated consumed carprofen dose was higher (49.08 mg/kg) as compared with the CarpTab group. Our previous study3 supports the observation of carprofen toxicity, as a high dose of carprofen leads to signs of agitation, restlessness, and impairment of analgesic efficacy with evidence of toxicity in NSG mice.3 Another study using C57Bl/6J mice has also been reported to show increased excitation of mice following oral intake of carprofen with drinking water at the 25-mg/kg/24-h dose, which was characterized by increased vocalization and tail elevation.31 The hypersensitivity observed in contralateral paws of the CarpGel-high and Carp25SC groups could also be attributed to this excitation of mice after carprofen administration as it was observed previously.31 Interestingly, in the current study, all carprofen groups, except CarpGel-high on D0 showed significant mechanical hypersensitivity reduction compared with the placebo group.
Thermal hypersensitivity was significantly reduced in the CarpTab group (on D1 and D2), the Carp25SC group (on D0, D1, and D2), and the CarpGel-low group (on D2) compared with the placebo group. This finding, along with our previous study,3 supports carprofen’s efficacy in attenuating thermal hypersensitivity better than the placebo group in adult NSG mice. However, none of the carprofen treatments were able to attenuate thermal hypersensitivity as compared with the baseline values. We attribute this to the possible need for a higher dose of analgesics to effectively reduce thermal hypersensitivity as reported in various studies.4,6,28,59 Furthermore, a study using male and female C57Bl/6J mice reported a lack of antinociceptive effect from carprofen administered either orally or subcutaneously (at 20 mg/kg) in the hot water tail immersion test compared with baseline values.31 Similar to our previous observation with the high-dose carprofen group,3 CarpGel-high showed no efficacy in reducing thermal hypersensitivity, which might be attributed to the development of toxicity.3
Although extensive research has been conducted on medication palatability in humans,17,30,48,70 there are only a limited number of studies in animals.2,5,81 Testing the palatability and acclimation of animals to the unmedicated gel or tablets is important to avoid neophobia of medicated gel or tablets especially if the gel is going to be used as a sole source of hydration or nutrition.11,45,47,57 Once the preferred gel is identified, acclimating the animal to the medicated gel is also essential, as the taste of the medication may alter the gel’s flavor and establish some level of plasma drug concentration before the intended surgical procedure. In the current study, we tested different nutritional and hydration gels before the start of the study (data not shown) and found that a bacon-flavored nutritional gel was most palatable to the NSG mice. Mice were acclimated to the carprofen-treated gels and tablets for 2 d before the surgery day.
Clinically effective carprofen plasma and tissue levels are not known in mice. However, the LD50 for oral carprofen has been reported as 282 mg/kg in mice and 149 mg/kg in rats.22 For other species, the hypothesized therapeutic plasma concentration of carprofen ranges from 20 to 24 μg/mL.49 A recent study using C57Bl/6J mice revealed that a supplement of carprofen via drinking water resulted in a peak plasma concentration at 24 h followed by a slight reduction and steady-state concentration over 5 d while maintaining the hypothetical therapeutic concentration.31 In the current study, carprofen plasma concentrations varied among all self-administered groups as it possibly depends on the time and amount of drug ingested by the mice voluntarily compared with our plasma collection times. During plasma analyses, HPLC-MS/MS had a carprofen detection limit of ≥0.6 μg/mL concentration, and some plasma samples displayed no peak values for concentrations <0.6 μg/mL. However, the hypothesized plasma concentration (20 to 24 μg/mL) was achieved in all self-administered oral carprofen groups during the average plasma concentration calculation, except for the CarpGel-low (at 4 h) and CarpGel-high (at 48 h) groups.
Despite carprofen’s efficacy in attenuating hypersensitivity in NSG mice, its use requires careful consideration due to potential toxicity. This is especially critical when carprofen is administered continuously at high doses, such as in self-administered setups. In the current study, fecal occult blood tests were used as a readout for drug toxicity. As such, 1 of 6 mice treated with CarpGel-high were positive for fecal occult tests on days 1 and 2. Fecal occult blood tests detect the presence of occult blood in the feces, which is indicative of GIT bleeding.3,36,43,44,58 The evidence of high-dose carprofen toxicity in GIT was also reported in previous studies.3,24,50,52,86 GIT bleeding is primarily caused by mucosal damage due to the inhibition of cyclooxygenase enzymes, leading to a reduction in protective prostaglandins.36,43,44,58 In addition to GIT toxicity, a high dose of carprofen causes impaired liver and platelet function and renal toxicity.3,24,50,52,82,86 Previous studies in mice have shown carprofen to be well tolerated when administered up to 20- to 25-mg/kg doses. For example, in C57Bl/6J mice, carprofen administered subcutaneously (at 20 mg/kg) or via drinking water (at 25 mg/kg/24 h) was well tolerated with no pathologic findings.31 In another study using CD-1 mice, carprofen administered subcutaneously at the 20-mg/kg dose had no incidence of gastric ulceration reported.44 Moreover, carprofen was well tolerated with no pathologic findings when administered subcutaneously at the 25-mg/kg dose in NSG mice.3 During self-administration of carprofen-medicated gel, tablet, or drinking water, continuous oral administration of carprofen may result in drug accumulation in the body due to insufficient clearance time between doses.20,22,40,65 This accumulation can increase toxicity resulting in the adverse effects mentioned above.
This study has several limitations. First, gel evaporation and loss of tablet fragments or moisture absorption of tablets in the cage pose challenges for accurate dose calculation. Second, mice may not consume enough carprofen-treated gels or tablets after a surgical procedure due to pain, potentially affecting the intended dosage. Third, this study is limited by an imbalance in sex distribution due to the limited availability of mice thus data analyses were performed by combining data from both sexes. Fourth, during estimation of the mean plasma concentrations, samples that had plasma carprofen concentration below 0.6 µg/mL were considered as zero because of detection limits. Fifth, in this study, distinct groups of mice were used for hypersensitivity assessment and plasma carprofen concentration analyses. Therefore, the plasma carprofen concentration may not precisely represent the levels in the hypersensitivity group, as surgical pain in that group could have led to a reduction in oral carprofen consumption. Sixth, this study did not include any tests to confirm the uniform distribution of the drug within the gel. This could be a contributing factor to the observed variation in plasma carprofen concentrations. Seventh, histopathologic assessment was not performed for carprofen toxicity. Eighth, in this study, tail handling of mice was used. To further minimize handling stress in mice, tunnel handling should be adopted.
Limited alternatives are available for analgesic medications in NSG mice. Among the commonly used analgesics in rodents, carprofen has been found to effectively reduce incisional pain in this strain. This study provides the first insights into the efficacy of 2 different oral carprofen formulations and points to the necessary precautions when preparing carprofen-treated gels and administering them orally. Due to the recommended dosing frequency, typically once or twice daily,3 the stress associated with repeated parenteral administration could be particularly harmful to immunocompromised animals such as the NSG strain. In this study, we evaluated the efficacy of carprofen-treated flavored nutritional gels and tablets. Our findings indicate that both CarpTab and Carp25SC effectively attenuate mechanical and thermal hypersensitivity, whereas CarpGel-low only effectively reduced mechanical hypersensitivity at the given carprofen dose used in the gel. These results support the use of orally administered CarpTab (for both thermal and mechanical sensitivity) and CarpGel-low (for mechanical sensitivity) as alternative analgesics to Carp25SC for incisional pain in NSG mice. Further studies are needed to determine the clinically effective dose of carprofen required in the gel to achieve consistent and clinically relevant plasma concentrations without compromising animal welfare due to toxicity. Furthermore, future research could focus on investigating potential sex-specific differences with equal distribution of sexes in the groups. From the results of this study, we recommend CarpTab as an alternative to Carp25SC for incisional pain procedures in NSG mice.
(A) Carprofen medicated nutritional gel diet (0.11 mg/mL and 0.22 mg/mL; NutraGel; BioServ) and (B) oral carprofen tablets (2 mg/tablet; Rodent MDs; BioServ).
Cage setup. (A) Carprofen-medicated gel diet and (B) oral carprofen tablets were placed on small weigh boats. To prevent contamination of the gel with bedding material, cup holders were used to keep the gel elevated and contained.
Experimental timeline. (A) Hypersensitivity testing. (B) Oral carprofen plasma concentration determination. (C) Fecal occult blood testing.
Mechanical hypersensitivity of ipsilateral (surgical) hind paw: measured as the number of paw withdrawals (mean ± SEM) of left hind paw in response to mechanical stimulus. Arrow indicates surgery on D0. *, Value is significantly different (P < 0.05) when compared with baseline (D-1) value of the same treatment group. #, Value is significantly different (P < 0.05) when compared with the placebo group at a specific time point for ipsilateral (surgical) and contralateral (control) groups.
Mechanical hypersensitivity of contralateral (control) hind paw: measured as the number of paw withdrawals (mean ± SEM) of right hind paw in response to mechanical stimulus. Arrow indicates surgery on D0. *, Value is significantly different (P < 0.05) when compared with baseline (D-1) value of the same treatment group.
Thermal hypersensitivity of ipsilateral (surgical) hind paw. Thermal latency measured in seconds (mean ± SEM) was defined as the response time of the left hind paw to thermal stimulus. Arrow indicates surgery on D0. *, Value is significantly different (P < 0.05) when compared with baseline (D-1) value of the same treatment group. #, Value is significantly different (P < 0.05) when compared with the placebo group at a specific time point.
Thermal hypersensitivity of contralateral (control) hind paw. Thermal latency measured in seconds (mean ± SEM) was defined as the response time of the right hind paw to thermal stimulus. Arrow indicates surgery on D0. *, Value is significantly different (P < 0.05) when compared with baseline (D-1) value of the same treatment group. #, Value is significantly different (P < 0.05) when compared between the carprofen groups at a specific time point.
Dose of carprofen consumed (mg/kg; mean ± SEM) by NSG mice over a 4-d period.
Plasma carprofen concentrations (μg/mL; mean ± SEM) in mice (n = 3/group/time point).
Contributor Notes