The induction of acute inflammation and associated hyperalgesia with carrageenan is one of the most widely used and well-established methods of studying acute inflammation in animal models, particularly mice and rats.1,2 Carrageenan is a sulphated mucopolysaccharide that is extracted from Irish moss (Chondrus crispus) and, when injected into mammalian (rodent) tissues, stimulates local inflammatory responses primarily through the aggregation of macrophages.3 Inflammation induced by carrageenan in rodent models is acute, nonimmune, and reproducible and has been valuable in inducing hyperalgesic conditions that can be subsequently utilized to assess effectiveness of analgesics.1,4 The tissue changes that occur exhibit the cardinal signs of inflammation (edema, hyperalgesia, and erythema) and develop immediately following subcutaneous injection in rats and mice.2 Carrageenan-induced inflammation has been used to study weight-bearing and behavioral changes associated with hyperalgesia in rats with significant findings.4,5 Utilization of carrageenan has been applied to avian models including cockerel and Hysex Brown domestic fowl chicks, assessing anatomical and physiological changes6 and alterations in withdrawal latencies.7 Basic protocols for carrageenan administration have been established,8 and the resolution of acute inflammatory changes following administration of anti-inflammatory analgesics in rodent models has previously provided humane use of this model in a laboratory setting.4
Alterations in weight-bearing ability have been shown to significantly differ in rats injected with carrageenan compared to control rats (injected with saline only).4 Similarly, use of a rotating perch or walkway, also referred to as a Rotarod, has been applied in rodent studies4,9 to evaluate locomotive abilities between treatment (carrageenan) and control animals. The rotational perch has also been used to assess locomotion in rodent studies10 to measure response to analgesics following carrageenan injection. Thermal hyperalgesia and a reduction in thermal threshold tolerance have been reported in both rats and mice injected with carrageenan in experimental settings.10–12 As footpad edema is a known sequela of carrageenan injection in rodents, this edema is commonly quantified via use of caliper measurements and has been shown to be a reliable way of measuring changes in injection site area in rats and mice.2
Although studies utilizing carrageenan to induce inflammation in avian models are limited, studies13–15 in which evaluations of weight-bearing perch load and locomotion while on a rotational perch have found significant differences in individual Hispaniolan Amazon parrots (Amazona ventralis) and green-cheeked conures (Pyrrhura molinae) experiencing induced acute arthritis. The bipedal avian anatomy, with weight-bearing placed solely on pelvic limbs, makes the assessment of weight-bearing load differential a reliable assessment for this study and has previously been used to assess lameness in psittacines and other avian species.13–16 The use of a thermal stimulus perch has been applied in a variety of avian studies17,18 and particularly in studies with cockatiels.19,20 In previous cockatiel studies19,20 utilizing the thermal perch, all birds were tested for nociception under unaltered conditions and cockatiels have shown to have reliable responses to this type of stimulus. Lastly, caliper measurements have been successfully used to measure other types of inflammatory lesions of the footpad in avian species such as penguins (Spheniscus magellanicus) and poultry (Gallus domesticus) when assessing for pododermatitis.21,22
Psittacine birds are common companion animals, and cockatiels are the most popular companion psittacine species.23 Psittacine birds are frequently presented to veterinarians in private practice, zoological collections, and rehabilitation facilities for injuries associated with soft tissue inflammation and lameness. Given their familiarity as a companion and in the laboratory setting, the cockatiel can be used as a model for other small psittacines and for studies of analgesics to evaluate pain control.
The objectives of this study were to evaluate a carrageenan-induced model of inflammation in the cockatiel and quantify the inflammatory changes and individual animal responses caused by this agent. Via use of the 4 described modalities, the following hypotheses were made: cockatiels injected with carrageenan will significantly exhibit unequal weight distribution on a weight-bearing load perch when compared to control birds, falter from a rotational perch of increasing speed sooner than control birds, have a lower thermal threshold when compared to control birds, and have the left footpad dimensions increase as a response to carrageenan injection when compared to both individual baseline measurements and control cockatiels.
Materials and Methods
Animals
Sixteen 3-year-old normal gray color morph (8 females and 8 males) cockatiels served as the study population. Birds were determined to be healthy based on results of a physical examination prior to the study. Cockatiels were housed individually in standard wire mesh laboratory cages (30.5 X 30.5 X 30.5 cm) with a wooden perch, hanging toy, feeder trough, and water line. Study birds were moved to 2 cage racks in a room separated from the breeding colony and acclimated for 30 days prior to study initiation to minimize effects of stress and minor variations in standard lighting and temperature conditions. Cockatiels were maintained on a day/night cycle of 12 hours of light and 12 hours of darkness, fed a commercial pelleted diet (Roudybush Inc) formulated for psittacines, and provided water ad libitum through the drip system. The room temperature was maintained between 22.2 and 22.8 °C and the humidity between 20% and 50%. This study was approved by the Institutional Animal Care and Use Committee at the University of California-Davis.
Induction of inflammation and allocation of study groups
Inflammation was experimentally induced in treatment cockatiels by unilateral subcutaneous injection of 0.05 mL of a 1% lambda carrageenan solution into the left footpad. The lambda carrageenan solution (TCI America) was prepared in a deionized water vehicle using published methods.8 A single 50-mL batch of 1% lambda carrageenan solution was used for all injection sessions, with individual aliquots of 1.0 mL being portioned out and stored at −62.2 °C until needed. On each day of carrageenan injection, a single aliquot was thawed to a liquid over a period of approximately 10 minutes by being held within a warm hand and then immediately drawn into individual 30-unit U100 insulin syringes (29-gauge needle) for injection.
Cockatiels were housed in 2 cage racks, and each set of 8 birds was randomly assigned as either treatment (carrageenan injection) or control (handling only, no injections) birds and balanced for sex and room location. Saline injection was omitted for control birds given the lack of significant changes appreciated from baseline measurements (weight-bearing, locomotion, and thermal hyperalgesia) in rodent carrageenan studies4,10 in which saline was injected in control animals. Treatments were performed between 6:50 and 8:10 AM on the first study day for each group. The cockatiels were manually restrained, and the left foot was then handled by the individual administering treatment. The left footpad of cockatiel was aseptically prepped and received either the placement of a capped needle along the plantar aspect of the footpad in control animals or injection of 0.05 mL 1% lambda carrageenan injection in treatment animals in the same location. Cockatiel handling and treatment required 1 to 2 minutes followed by return to their cages.
The individual obtaining measurements and photos (NAM) was masked to treatment assignment. Following completion of thermal perch measurements, at approximately 36 hours posttreatment, all cockatiels received a course of parenteral meloxicam (Metacam) 2 mg/kg IM every 12 hours for 7 days and an additional 7 days of meloxicam 2 mg/kg SC every 12 hours for provision of analgesia and anti-inflammatory therapy. As additional measurements (weight-bearing perch, rotating perch, and footpad dimension) were obtained following the thermal perch measurements, meloxicam administration was implemented to provide pain management and maintain appropriate animal welfare during ongoing data collection. All study cockatiels were administered meloxicam to keep all variables (apart from treatment) as constant as possible between groups.
Weight-bearing load perch
A modified incapacitance meter utilized in previous psittacine studies13–15 was used to assess weight-bearing load capacity for all cockatiels in this study. Standard rodent footpads were converted into a divided perch so that the extent of weight bearing could be measured independently for each foot. A black, 30.5 X 21.6 X 12.7-cm plexiglass box with a transparent front and hinged back door was placed around the perch to limit movement by the cockatiels during assessment. The perch was seated approximately 15.2 cm from the platform base to prevent any cockatiel’s tail from contacting it and potentially altering measurement readings. The perch used dual-channel weight averaging, which enabled testing of both pelvic limbs simultaneously and recorded the mean weight in grams during a 10-second interval. A total of 4 consecutive 10-second interval measurements were obtained for each cockatiel at each assessment point throughout the study. An accurate measurement was described and included when a cockatiel remained calm during the 10-second interval and with one foot on each perch bar (or with the right foot firmly on the perch bar for birds that became non–weight-bearing on their injected left limb at any point during the study). If a cockatiel exhibited any abrupt movements (spinning on the perch, biting the perch, transitioning to only one side of the perch, rapid head-bobbing, rousing, or preening), then the measurement was discarded and a new measurement was obtained once the cockatiel returned to a more stationary state. A small video camera (Logitech Pro Webcam) was placed in front of the box allowed behavioral responses to be remotely monitored in real time and uninfluenced by the present of the observer (NAM) in the room. A 1-month acclimation period was used to condition the cockatiels to perch inside the test box and remain calm within the box for 4- to 5-minute intervals. A mock study day, 1 week prior to carrageenan administration, was used to establish baseline weight-bearing load performance. Individual baseline measurements were obtained for each cockatiel the morning of injection. Following administration of treatments, weight-bearing load perch measurements were collected at 1.5, 3, 4.5, 6, 7.5, 9, 24, 47, 71, 96, 120, 143, 167, and 336 hours. Data from each cockatiel at each time point were calculated to reflect the difference in extent of weight bearing between the carrageenan-injected (left) limb and the noninjected (right) limb by using the following equation: DBW = absolute value ([weight placed on unaffected right limb – weight placed on affected left limb]/[weight placed on right limb + weight placed on left limb]) X 100. A DBW of 0% corresponded to a bird that bore weight equally on both pelvic limbs, whereas a DBW of 100% indicated that the bird bore no weight on its affected left limb.16
Rotational perch
A rotating perch utilized in previous psittacine studies13,24 was used to evaluate locomotive abilities and pelvic limb dexterity. The perch was constructed by use of a 11.4-cm-long perch with a diameter of 3.2 cm and was fixed within a black 50.8 X 45.7 X 14.0-cm plastic box with a transparent front and hinged back door. Perch rotation was produced by a direct current gear head and motor combination attached to a variable-speed control box. Rotation of the perching rod encouraged each cockatiel to actively walk at their normal ambulatory speed while the perch turned. A video camera in front of the box allowed behavioral responses to be remotely monitored in real time and uninfluenced by the present of the observer (NAM) in the room. For each rotating perch measurement, the cockatiel had 10 seconds to settle onto the perch before it began rotating. Rotational velocity was increased 10 ± 1 rotations/minute every 10 seconds until a maximum speed of 30 rotations per minute was reached. This maximum speed was then maintained for a total duration of 120 seconds or until the cockatiel faltered off the perch, whichever occurred first. A 1-month acclimation period was used to condition the cockatiels to the rotating perch for 4- to 5-minute intervals. A mock study day to establish baseline rotating perch performance was completed 1 week prior to carrageenan administration, and individual baseline measurements were obtained for each cockatiel prior to study initiation. Following administration of treatments, rotating perch performance measurements were collected at 9, 24, 47, 71, and 96 hours. The change in rotational perch time from baseline was used to compare groups.
Thermal stimulus perch
Foot withdrawal responses to a thermal stimulus were measured by placing each cockatiel in a test box measuring 31.8 X 40.6 X 14.0 cm and equipped with a thermal stimulus perch. The perch was located approximately 11.4 cm from the transparent front of the box and 26.7 cm from the hinged back door of the box. The box also contained a gradual downward plastic ramp that the cockatiels could use to climb onto the perch themselves. The test box had dark, nonreflective plastic sides and a clear front with a video camera placed in front of the box for remotely monitoring birds in real time, uninfluenced by the present of the observer (NAM). All thermal withdrawal responses were measured by 1 observer (NAM) who was masked to the treatments. The perch contained a metal stimulus bar that delivered a gradually increasing (∼0.34 °C/sec) thermal stimulus to the plantar surface of the left foot of each bird. The temperature range (37 to 56 °C) for the thermal stimulus was restricted to avoid thermal damage to the foot. A cockatiel was able to escape the brief noxious thermal stimulus by lifting or rapidly shuffling its left foot (withdrawal response) from the metal stimulus bar. When the withdrawal response was witnessed, the observer pressed the deactivation button on the apparatus to rotate the metal stimulus bar 180° to the underside of the perch, providing an unheated perch surface for cockatiels to place the left foot back on the perch within 1 to 2 seconds thereafter. The thermal withdrawal threshold was defined as the perch temperature concomitant with the observed left foot withdrawal response. A 1-month acclimation period prior to treatments was used to condition cockatiels to the inactive thermal perch (no thermal stimulus applied) initially and then the active thermal perch (thermal stimulus applied), conditioning the birds to remain in the box for 2- to 3-minute intervals. A mock study day to establish baseline thermal threshold performance for each cockatiel was carried out 1 week prior to carrageenan administration as well. Thermal stimulus withdrawal testing took place the day following treatments, with measurements being taken at time “0” (baseline; 24 hours posttreatment), 24.5, 25.5, 27, and 30 hours posttreatment.
Imaging of footpads and footpad swelling grading
Objective assessment of the left footpad of each cockatiel used sequential images of the left footpad evaluated for vertical and horizontal lengths (both in mm), with an increase in footpad dimensions associated with swelling. Vertical length was defined as the distance between the interdigital space of digits 2 and 3 and the interdigital space of digits 1 and 4. Horizontal length was defined as the distance between the medial most aspect of digit 1 to the lateralmost aspect of digit 4. Footpad lesion area was then calculated through multiplication of the horizontal length with the vertical length. For each image collected using an external camera (Samsung Galaxy S6), the cockatiel was manually restrained in dorsal recumbency. A padded glass frame measuring 10.2 X 15.2 cm was gently placed along the plantar aspect of both footpads to prompt each cockatiel to exert an opposing force against the glass and extend the digits to provide an open view of the footpad of each foot. A 10-cm ruler was permanently attached to the lower aspect of the glass frame to serve as a reference for subsequent digital caliper measurements. A 2-week acclimation period was used to condition the cockatiels to the positioning needed for these images and the tactile experience of having the glass frame pressed against their feet. Baseline images of each cockatiel’s footpad were taken approximately 12 hours prior to treatment and 9, 24, 47, 71, 96, 120, 143, 167, 336, 504, 672, 1,008, 1,344, and 2,016 hours posttreatment. Images taken were then analyzed using ImageJ commercial software. For each image, the appropriate scaling was set using the 10-cm ruler as a reference to ensure uniform scaling between images prior to footpad measurements using the digital caliper in the software. Grading were also made by a single observer (NAM) regarding the gross appearance of each cockatiel’s left footpad and whether it appeared swollen compared to that of the right (graded on a subjective scale of 0 = none, 1 = mild, 2 = moderate, and 3 = severe).
Statistical analysis
Outcome variables were analyzed using linear mixed models with their baseline, treatment, time, treatment X time interaction, and sex as fixed effects and individual cockatiels as random effect. For the left foot weight loading, the right foot weight loading was added as a fixed effect to assess contralateral weight shifting. Normality and homoscedasticity of the residuals as well as the presence of outliers were checked on quantile residual plot and standardized Pearson residual plots. An ANOVA was performed on the fixed effects. Post hoc analysis was performed using a Tukey adjustment and using time as an ordinal categorical variable to be able to compare between individual time points.
An ordinal logit mixed model was used to investigate the inflammation score using individual cockatiels as random effect and time and treatment as fixed effects. Mixed models were performed using R-package “nlme” and plots were generated with R-package “ggplot2.”25 An alpha of 0.05 was used for statistical significance and R (version 4.0.4, R Foundation for Statistical Computing).
Results
Weight-bearing load perch
The left foot weight loading (measured in grams) was significantly associated with the interaction of treatment X time (P < 0.001), right foot weight loading (P < 0.001), baseline values (P < 0.001), but not sex (P = 0.062). For the post hoc analysis on time when it was used as a categorical variable, a model with a heterogeneous variance over treatment was found to better fit the data (P < 0.001). When compared to control, treated birds had a significant decrease in their left foot weight loading at 143 hours (P = 0.016) and 167 hours (P = 0.034) (Figure 1). When compared to baseline, there was no significant difference over time in the control group (all P > 0.05). However, in the treatment group, the left foot weight loading was significantly decreased from baseline at 120, 143, and 167 hours (P = 0.013, P = 0.002, P = 0.006, respectively). For the baseline, every 1 increase in baseline led to a corresponding 0.78 ± 0.16 increase in the left foot weight loading. For the right foot, the decrease in weight loading of the left foot corresponding almost exactly to an increase in weight loading at 0.98 ± 0.01.
Differences between treatment (n = 8; dashed line) and control (8; solid line) groups in left foot weight-bearing load over time. Weight-bearing measurements (in grams) obtained beginning at time “0” immediately prior to subcutaneous injection of 0.05 mL of a l% lambda carrageenan solution into the left footpad in treatment birds or handling alone in control birds and continuing up to 336 hours posttreatment. The left foot weight loading (measured in grams) was significantly associated with the interaction of treatment X time (P < 0.001), opposing right foot weight loading (P < 0.001), and baseline values (P < 0.001). Error bars are means ± SE. Smaller graph illustrative of first 24 hours postinjection.
Citation: American Journal of Veterinary Research 83, 8; 10.2460/ajvr.22.02.0020
For the percent DBW, regular linear mixed models did not comply with assumptions of normality of the residuals even after log transformation and altering the covariance matrix structure of the mixed model. Therefore, a rank transformation was applied to fit the assumptions. Treatment was associated with higher DBW than control birds (P = 0.002) with a rank difference of 60 (median treatment, 9.2%; median control, 3.4%).
Rotational perch
For the rotating perch, almost all birds in each treatment and all time points had the same value, so no significant differences were observed for any time point (all P > 0.05). For the vast majority of all time points, all 16 cockatiels remained in the rotating perch for the full 120 seconds at the maximum 30 rotations per minute speed. A single female treatment bird faltered from the rotating perch once after 106 seconds at the 30 rotations per minute speed during the 24-hour posttreatment timepoint but then remained stable on the perch for the maximum 120 seconds for all subsequent time points.
Thermal stimulus perch
For the thermal withholding threshold, only baseline had a significant effect at 0.4 ± 0.1 °C for each 1 °C increase in baseline temperature (P = 0.01). No significant effect of time or treatment was seen (all P > 0.05) (Figure 2). Mean standard deviation over time within control cockatiels was 0.69 °C, indicating low variability and reliable performance of these birds on the thermal stimulus perch.
Lineplot of the changes in thermal threshold withdrawal over time and treatment in comparing treatment (n = 8) to control (8) groups. Treatment birds received subcutaneous injection of 0.05 mL of a l% lambda carrageenan solution into the left footpad while control birds received handling only. For each cockatiel, thermal threshold was determined at time “0” (∼24 hours posttreatment), 24.5, 25.5, 27, and 30 hours posttreatment. No significant effect of time or treatment was seen (all P > 0.05). Error bars are means ± SE.
Citation: American Journal of Veterinary Research 83, 8; 10.2460/ajvr.22.02.0020
Imaging of footpads and footpad swelling grading
For the vertical size, only treatment (P = 0.018) and baseline values (P = 0.002) had significant effects. The treatment was associated with a significant increase in vertical size of 0.96 ± 0.25 mm compared to control and the baseline increased that measurement by 0.47 ± 0.13 mm for each 1-mm increase in baseline. Similar results were obtained for horizontal with only treatment (P < 0.001) and baseline values (P = 0.006) having a significant effect. The treatment was associated with a significant increase in horizontal size of 1.30 ± 0.19 mm compared to control and the baseline increased that measurement by 0.41 ± 0.13 mm for each 1-mm increase in baseline. Similar results were obtained for horizontal size with only treatment (P < 0.001) and baseline values (P = 0.006) having a significant effect.
For lesion area, there was a significant time X treatment interaction effect (P = 0.02) as well as baseline values (P = 0.004). When compared to control, treated birds had a significant increase in their left foot lesion area at from 47 hours to 504 hours (all P < 0.05) (Figure 3). When compared to baseline, there was no significant difference over time in the control group (all P > 0.05). However, in the treatment group, the left foot lesion area was significantly increased from baseline from 96 hours to 167 hours (all P < 0.05). For the baseline, every 1 increase in baseline led to a corresponding 0.33 ± 0.11-mm2 increase in the left foot lesion area.
Lineplot of the changes in left footpad area (in mm2) over time and treatment. Baseline footpad measurements obtained beginning at time “0” prior to subcutaneous injection of 0.05 mL of a l% lambda carrageenan solution into the left footpad in treatment birds (n = 8) or handling alone in control birds (8) and with subsequent footpad measurements continuing up to 84 days posttreatment. For left footpad area, there was a significant time X treatment interaction effect (P = 0.02) as well as baseline values (P = 0.004). When compared to control, treated birds had a significant decrease in their left foot lesion area at from 47 to 504 hours (all P > 0.05). When compared to baseline, there was no significant difference over time in the control group (all P > 0.05). However, in the treatment group, the left foot lesion area was significantly increased from 96 hours to 167 hours (all P < 0.05). Error bars are means ± SE.
Citation: American Journal of Veterinary Research 83, 8; 10.2460/ajvr.22.02.0020
For the swelling grade, the ordinal logit mixed model did not converge and a linear mixed model on rank transformed data was performed instead. Treatment was associated with significantly higher swelling grade (P < 0.001). The foot pad lesion significantly decreased weight bearing on that leg by 1 for each 0.54 mm2 (P < 0.001).
Discussion
Subcutaneous injection of carrageenan into the left footpad of cockatiels prompted the formation of inflammation and altered weight-bearing of the injected limb, as has similarly been reported in rodent studies.4,5,8 The majority of cockatiels treated with carrageenan developed changes in weight-bearing load, differing significantly from control cockatiels as evidenced by both their weight-bearing perch performance and changes in their percent DBW. Interestingly, some treatment cockatiels exhibited weight-bearing changes as early as 1.5 hours postinjection while others did not develop measurable differences until the 96-hour time point. This makes prediction of peak effects difficult in the cockatiel, in contrast to the established 3- to 4-hour postinjection peak in rodent models4,5 and the 2-hour postinjection peak in chick models.6,7 This lack of consistency in timing of peak effects may be due to the reduced elasticity and scaled skin of adult cockatiels compared to that of rodents and immature poultry, individual variation in inflammatory response to the novel carrageenan injection between cockatiels, or slight variations in depth of the needle into the footpad.
The lack of thermal threshold changes in the carrageenan-treated cockatiels was unexpected. Since the majority of carrageenan-injected cockatiels displayed alterations in their weight-bearing ability off of the injected left limb during the course of the study, it would be reasonable to assume hyperalgesia was associated with that foot. However, the presumptive hyperalgesia did not translate to a lower thermal threshold for carrageenan-injected cockatiels when placed on the thermal perch. This may be due to a variety of factors, particularly given that significant differences in weight-bearing load between treatment and control cockatiels did not occur until later in the study (143 and 167 hours postinjection). As thermal threshold was assessed 24 to 30 hours postinjection, this window may have been too early to catch hyperalgesic changes that could have accompanied the later changed in weight-bearing load. Alternatively, assessing thermal threshold at a timepoint earlier than the 24-hour mark (such as 12 hours postinjection) may have allowed for capture of acute inflammation and associated hyperalgesia that could have resulted in thermal threshold changes between treatment and control cockatiels. Of note, studies19,20 assessing for a potential increase in thermal threshold secondary to administration of analgesics (hydromorphone and buprenorphine, respectively) in cockatiels have similarly resulted in no significant difference at the doses evaluated between the thermal thresholds of control versus treatment groups. For this species, it may simply be that thermal nociception is an insensitive modality for assessing hyperalgesia of the foot. Modalities such as von Frey filaments that use external force directly applied to the footpad may be an alternative method to assess for hyperalgesia in future studies.
The treatment cockatiels’ locomotive capabilities on the rotating perch were not significantly different from baseline assessment or when compared to control birds. In a previous study13 with Hispaniolan Amazon parrots with experimentally induced arthritis, there was a significant difference between baseline rotating perch performance and subsequent performance postinjection; however, the differences between groups (those treated with varying doses of meloxicam for analgesia vs control birds given saline) did not reach statistical significance. In comparing the methodology of the study of Cole et al13 in 2009 to this study, the rotating perch speed in the study of Cole et al was increased by 11.6 ± 1.0 rotations every 10 seconds until a parrot could no longer maintain walking on the perch (time on perch not capped), as opposed to this study in which rotation speed did not exceed 30 rotations per minute and there was a set maximum perch time of 120 seconds. Had the rotation speed continued to be increased every 10 seconds, it is possible that a difference between control and treatment cockatiels may have become apparent. In similar carrageenan studies9 performed in rats, a significant difference between control and treatment animals was likewise absent when assessing locomotive performance via a Rotarod. Compensation with the opposite pelvic limb and/or wings (or the unaffected 3 limbs in rodents) may allow animals to maintain locomotive capabilities when solely affected by soft tissue inflammation, as opposed to the articular involvement in the study of Cole et al.13
Sequential footpad imaging of the cockatiel feet and subsequent vertical and horizontal measurements proved to be a reliable means of evaluating the swelling associated with inflammation caused by carrageenan treatment. Each cockatiel served as its own baseline reference when comparing individual as well as group measurements. Uniformly, treatment cockatiels had an increase in both the vertical and the horizontal dimensions of their injected left footpad. These findings are consistent with the edema reported in both chick and rodent models.6,8 The cockatiel footpad dimensions were collected for 84 days (12 weeks) postinjection in contrast to the 1 to 2 days of data collection in typical rodent studies2 and the carrageenan study of Ito et al6 in 1989 in chicks. This long-term follow-up in the current study provided critical information regarding acute and chronic changes of the footpad size associated with carrageenan injection. The carrageenan-injected footpads remained significantly (P < 0.05) greater than the footpad of control birds for the first 3 of the 12-week follow-up period postinjection. Additionally, the swelling continued to worsen for some treatment cockatiels over the first week postinjection. Although all cockatiels were measurably sound as evidenced by return to normal weight-bearing ability within 2 weeks postinjection, footpad dimensions did not return to baseline for any of the treatment cockatiels. It is hypothesized that the residual footpad changes are likely underlying scarring/fibrotic changes secondary to the distortion caused by the initial edema and tension placed on the scaled skin of the cockatiels’ footpads. In unpublished data evaluating histological changes associated with carrageenan injection in cockatiels, the inflammatory effects did not extend past the level of the skin and subcutaneous tissues when assessed 3 months postinjection, with panniculitis being the only observed alteration to the injected footpad. The chronic changes in increased footpad size warrant further investigation and should be taken into consideration in future studies for inflammation and response to treatment with anti-inflammatory or analgesic agents. Cockatiels used for this study may not be appropriate for use in future studies similarly evaluating inflammatory processes of the foot or pelvic limb, as hyperalgesic priming has been documented in rats that have undergone carrageenan administration via injection.26
An important limitation for this study was the small sample size, which constrained the authors from making strong conclusions for the use of carrageenan footpad injections as a novel model of inflammation in the cockatiel without further investigation. A small sample size could additionally have contributed to the presence of type II error, particularly given the level of individual variability present during several aspects of this study. Time to onset of peak inflammation, for example, based on weight-bearing perch measurements and footpad dimensions, was more variable than reported in rodent carrageenan models of inflammation. However, both similar and smaller sample sizes have been used to evaluate weight-bearing and locomotive abilities and thermal thresholds in psittacines and yielded significant results.13,18
The ability to position each cockatiel in the same position during image capture of the footpads had limitations. Performing measurements in conscious cockatiels without sedation reduced handling time and allowed to identify significant differences in this study; however, small differences in positioning, individual birds’ opposing footpad pressure applied to the glass frame, and tolerance to handling when obtaining images made collection of the data challenging. Were this portion of the study to be repeated or pursued in a different avian species, a more uniform method of measuring changes in footpad size/volume like plethysmometry may produce more accurate results as shown in rodents.2
In choosing the concentration and amount of lambda carrageenan in which to administer to the cockatiels in this study, decisions were made in combination with both pilot study results not presented in this study and doses described in previous chick and rodent studies.6–8 Saline injection was omitted for control birds given the lack of significant changes appreciated from baseline measurements (weight-bearing, locomotion, and thermal hyperalgesia) in rodent carrageenan studies in which saline was injected in control animals.4,10 In terms of causing inflammatory changes to the injected footpad, all 8 cockatiels injected with carrageenan developed some level of grossly appreciable response. However, not all injected cockatiels subsequently exhibited any changes in terms of their weight-bearing distribution or other measured parameters. In studies evaluating carrageenan injections in chick models, there have similarly been mixed findings in the overall level of response.6,7 Findings described by Roach et al7 in 2003 showed changes in withdrawal latency and foot volume to be concentration dependent in injected animals (concentrations ranged from 0% to 1% assessed, but volume given not described). In contrast, Ito et al6 in 1989 tested carrageenan concentrations from 0.2% to 1% (0.1-mL injection volume) and did not find results to be as predictable; the 0.5% concentration produced the most overt results in terms of increased foot volume while the 0.2% and 1% results were described as being “not significantly altered” from baseline values. Given the scaled skin anatomy and reduced skin elasticity of the avian pelvic limb and foot, this may additionally make it difficult to determine the dosage response and extent of inflammation resulting from carrageenan injection in birds compared to rodent models.
Pursuit of this carrageenan model was intended to mimic inflammatory conditions appreciated in certain scenarios such as lameness and soft tissue trauma that are commonly presented in clinical settings. Similarly, the cockatiel was chosen as an ideal study species given this animal’s familiarity as a popular companion animal within psittacine species.23 However, it is established that anatomical differences and their associated responses to inflammation can vary even between psittacine species, let alone between different avian orders. As the foot is a crucial tool in food consumption for larger psittacines (gripping of food items) and raptor species (penetration of prey items and subsequent grasping of flesh), it is possible that these types of birds would have additional measurable effects (such as decreased grip strength and more prominent weight loss) as a result of carrageenan injection. Were this model to be further explored in future, administration of carrageenan in other psittacine species (both large and small) would be helpful in determining if similar inflammatory responses are elicited postinjection. Potential alterations in carrageenan-induced inflammatory patterns due to conformational differences between digital anatomy (zygodactyl vs anisodactyl vs tridactyl, etc) and the presence of additional distal limb features such as interdigital webbing (Anseriformes, Gruiformes, Sphenisciformes, etc) would also be intriguing to investigate.
Performance of this study allowed for the development and evaluation of a novel method of inducing inflammation via use of lambda carrageenan in cockatiels. Assessment of weight-bearing load and footpad dimensions was sensitive and reliable assessments of inflammation and differentiating treatment from control cockatiels. In contrast, thermal threshold withdrawal and rotational perch locomotion were not found to significantly differ between groups, despite being reliable methods. Cockatiels’ weight-bearing load returned to baseline values within 2 weeks; however, left footpad dimensions did not return to baseline. The carrageenan-induced inflammatory model provides an additional means of evaluating inflammation and lameness in small psittacine species.
Acknowledgments
The authors of this study thank the Center for Companion Animal Health of the University of California-Davis School of Veterinary Medicine and the Richard M. Schubot Parrot Wellness and Welfare Program for grant support of this research endeavor.
The authors declare that there were no conflicts of interest.
A special thanks is extended to Kristy Portillo, Daniel Pichardo, and Alexis Adams-Lucas for their direct support with husbandry and handling of the cockatiels throughout this project.
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