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Published in final edited form as: Exp Neurol. 2025 Oct 21;396:115522. doi: 10.1016/j.expneurol.2025.115522 Search in PMC Search in PubMed View in NLM Catalog Add to search Liposome-encapsulated clodronate and COX-2 inhibitor treatment impair ventilatory recovery but improve compensatory locomotor function following cervical spinal cord injury in rats Aaron L Silverstein Aaron L Silverstein A Department of Neuroscience, Spinal Cord and Brain Injury Research Center, College of Medicine, University of Kentucky, 741 S. Limestone St., Lexington, KY, 40508 Find articles by Aaron L Silverstein A, * , Christopher M Calulot Christopher M Calulot A Department of Neuroscience, Spinal Cord and Brain Injury Research Center, College of Medicine, University of Kentucky, 741 S. Limestone St., Lexington, KY, 40508 Find articles by Christopher M Calulot A , Christopher J McLouth Christopher J McLouth B Department of Biostatistics, College of Public Health, Biostatistics Consulting and Interdisciplinary Research Collaboration Lab, University of Kentucky, 725 Rose St. Lexington, KY, 40536 Find articles by Christopher J McLouth B , John C Gensel John C Gensel C Department of Physiology, Spinal Cord and Brain Injury Research Center, College of Medicine, University of Kentucky, 741 S. Limestone St., Lexington, KY, 40508 Find articles by John C Gensel C , Warren J Alilain Warren J Alilain A Department of Neuroscience, Spinal Cord and Brain Injury Research Center, College of Medicine, University of Kentucky, 741 S. Limestone St., Lexington, KY, 40508 Find articles by Warren J Alilain A, * Author information Article notes Copyright and License information A Department of Neuroscience, Spinal Cord and Brain Injury Research Center, College of Medicine, University of Kentucky, 741 S. Limestone St., Lexington, KY, 40508 B Department of Biostatistics, College of Public Health, Biostatistics Consulting and Interdisciplinary Research Collaboration Lab, University of Kentucky, 725 Rose St. Lexington, KY, 40536 C Department of Physiology, Spinal Cord and Brain Injury Research Center, College of Medicine, University of Kentucky, 741 S. Limestone St., Lexington, KY, 40508 7. Author Contribution Statement Aaron Silverstein: Conceptualization, Methodology, Software, Formal analysis, Investigation, Data Curation, Writing- Original Draft, Writing- Review & Editing, Supervision, Visualization & Funding Acquisition. Christopher Calulot: Methodology, Investigation, Writing- Review & Editing. Christopher McLouth: Software, Formal analysis, Resources, Writing- Original Draft, Writing- Review & Editing. John Gensel: Conceptualization, Methodology, Resources, Writing- Review & Editing, Visualization, Supervision, Project Administration, & Funding Acquisition. Warren Alilain: Conceptualization, Methodology, Resources, Writing- Review & Editing, Visualization, Supervision, Project Administration, & Funding Acquisition. * Corresponding authors: Warren J. Alilain, Ph.D., Professor, B469 BBSRB, 741 S. Limestone Avenue, Lexington, KY 40536, [email protected] , Aaron L. Silverstein, Ph.D., MD/PhD Candidate, MS-3, B431 BBSRB, 741 S. Limestone Avenue, Lexington, KY 40536, [email protected] Issue date 2026 Feb. PMC Copyright notice PMCID: PMC12582558 NIHMSID: NIHMS2119318 PMID: 41130383 The publisher's version of this article is available at Exp Neurol Abstract Over half of all spinal cord injuries (SCIs) in the United States occur at the cervical level and can cause locomotor deficits and life-threatening breathing dysfunction. Interestingly, the bisphosphonate drug clodronate has shown efficacy in ameliorating tissue damage and improving locomotor recovery acutely after experimentally induced thoracic SCI. Thus, we hypothesized that clodronate treatment would improve recovery of breathing and locomotor function following a C2 hemisection (C2Hx) model of cervical SCI in rats. Serendipitously, changes to animal use guidelines led to the inclusion of carprofen, a non-steroidal anti-inflammatory drug (NSAID), as another independent variable in our study. We treated adult rats intravenously with either liposomal clodronate or saline via the tail vein at days 1, 3, and 6 post-C2Hx. Carprofen treatment was administered subcutaneously on days 0, 1, and 2 post-injury. We used whole-body plethysmography to measure ventilatory function and the semi-automated CatWalk ® gait analysis system to assess locomotor function through 4 weeks post-SCI. Contrary to our initial hypothesis, we found that both liposomally encapsulated clodronate and carprofen impaired ventilatory recovery following C2Hx. However, in alignment with our hypothesis, clodronate improved locomotor function on the side contralateral to injury. To reconcile these seemingly conflicting outcomes, we propose that clodronate treatment may exacerbate lung inflammation, altering peripheral-to-central modulation of respiratory output—highlighting that the effects of these treatments may be specific to injury level and target organ system. By further elucidating clodronate and carprofen as clinically relevant therapeutics, the work described here serves to advance efforts to improve care for individuals living with SCI. Keywords: Spinal cord injury, NSAIDs, clodronate, macrophage depletion, breathing, locomotor function 1. Introduction Spinal cord injury (SCI) poses a significant public health concern that currently affects approximately 300,000 individuals within the United States 1 . SCI has an annual incidence of 18,000 and injuries most often occur at the cervical level 1 . High cervical SCIs not only affect individuals’ ability to walk independently, but can cause life-threatening respiratory motor paralysis or paresis 1 , 2 . Indeed, the bulbospinal innervation to the phrenic motor pool is located in the cervical spinal cord and controls the diaphragm, the primary inspiratory muscle in mammals 3 . Thus, cervical SCI commonly leads to breathing dysfunction or even cessation necessitating mechanical ventilation. Despite the serious impact that high cervical SCI has upon health and quality of life, no clinically successful treatment is available to reverse injury-induced loss of breathing function in humans 4 , 5 . While many novel pre-clinical treatments show promise, the process of establishing both their safety and efficacy in clinical trials has largely been unsuccessful 5 . However, the drug clodronate, a member of the clinically-utilized bisphosphonate drug class, has demonstrated efficacy in improving locomotor recovery and tissue sparing in addition to diminishing inflammatory signaling, axonal dieback, and lesion extent in animals following experimentally-induced caudal cervical (C8), thoracic-level, or ischemic SCI 6 – 14 . Systemic treatment of murine subjects with liposomal formulations of clodronate depletes populations of hematogenous monocytes which differentiate to become monocyte-derived macrophages (MDMs) within the injured spinal cord, thus ameliorating their contribution to secondary inflammatory processes following SCI 6 – 14 . Bisphosphonate drugs like clodronate are already utilized for clinical treatment of conditions affecting phagocytosis-mediated bone resorption such as osteoporosis, various skeletal and immune malignancies, and complications secondary to malignancy 15 . As a result, the repurposing of such a drug for clinical treatment of SCI might meet with greater success than for other therapeutics which have not yet been approved for clinical use of any kind, a highly valuable approach in the study of more effective translational science 16 . However, it is yet unknown whether clodronate treatment will improve breathing and locomotor recovery after high cervical SCI. Thus, we hypothesized that systemic treatment with liposomal clodronate would improve breathing and locomotor recovery following the C2 hemisection (C2Hx) model of cervical SCI in rats. Serendipitously, the common post-surgical NSAID drug carprofen became a secondary therapeutic intervention included within this study ( Figure 1 ), due to changes made to animal care protocols during ongoing experimentation. Carprofen is a preferential cyclooxygenase-2 (COX-2) inhibitor and non-steroidal anti-inflammatory drug (NSAID), a variety of which have been shown to diminish oxidative stress, decrease markers of neuroinflammation, play a neuroprotective role, ameliorate motor deficits, and prevent complications following SCI 17 – 21 . As a result, we additionally hypothesized that carprofen would improve functional breathing and locomotor recovery either alone or in combination with clodronate in comparison to control. Figure 1: A graphical depiction of the treatment groups for this experiment. Open in a new tab As originally designed, we were to investigate the differential effects of clodronate-mediated macrophage depletion or control treatment on the recovery of breathing and locomotor function after cervical spinal cord injury. However, upon the serendipitous addition of carprofen as an independent variable we split those original 2 treatment groups, giving rise to the final four groups treated with clodronate, clodronate + carprofen, saline, or saline + carprofen. All sample sizes are listed for each treatment and timepoint cohort. Importantly, the number of subjects lost to attrition are described within parentheses. 2. Materials and Methods 2.1. Animal subjects All animal care and handling were carried out in compliance with the Institutional Animal Care and Use Committee guidelines at the University of Kentucky (Protocol 2020–3704). Subjects were split into two groups with terminal endpoints planned at 1-week and 4-weeks post-injury. A total of 93 female, retired breeder, Sprague Dawley ® outbred rats (Inotiv, West Lafayette, IN; pre-injury weight range 255–380g) were used for experimentation across both the 1-week (n = 18) and 4-week (n = 75) timepoints ( Figure 1 ) and were 10–14 months of age at time of experimentation, housed on a 12/12 light/dark cycle, and fed normal chow diet ad libitum . 16 of these subjects were lost to attrition with 9/16 having received at least one of their clodronate or saline tail vein injections and all carprofen injections where applicable prior to demise ( Figure 1 ). Hereafter, all referenced group sizes and analyses omit subjects lost to attrition. All subjects were randomized with respect to treatment with clodronate or saline, but only 29/61 carprofen-exposed subjects were randomized with respect to carprofen due to its serendipitous addition to the current experiment ( Figure 1 ). 2.2. Surgical procedure and aftercare for C2 hemisection A C2 hemisection was performed on isoflurane anesthesia according to previously described procedure 22 , 23 . Briefly, the C2 spinal cord was posteriorly exposed and a 27-gauge needle used to complete the left-sided hemisection. Bupivacaine hydrochloride with epinephrine was instilled along the incision during closing, and both subcutaneous buprenorphine and saline were administered during initial anesthesia recovery, with buprenorphine administration repeated once more the following day for a total of two doses. Post-operative care included close wellness and weight monitoring and supplemental saline administration with wound clip removal at 7–10 days post-injury. 2.3. Therapeutic drugs and administration The veterinary NSAID carprofen (Covetrus, Portland, ME, USA) was administered subcutaneously to unanesthetized subjects in 3 doses of 1 mL each at a 1.5 mg/mL concentration immediately following surgery and once a day for the subsequent 2 days for a subset of animals post-surgery as depicted in Figure 2A – B 24 , 25 . Subjects not treated with carprofen were not administered any control injection. One subject assigned to the saline group was inadvertently exposed to carprofen on day 2 post-injury but post-hoc outlier analysis failed to justify exclusion of this subject’s data from further analysis. Figure 2. Depiction of experimental paradigms and plethysmography protocols. Open in a new tab According to treatment group assignments, subjects across both timepoints received subcutaneous carprofen injections at days 0, 1, and 2 post-injury while clodronate or saline control was administered intravenously via the tail vein on days 1, 3, and 6 post-injury. Whole-body plethysmography (WBP) and CatWalk locomotor assessments were performed prior to C2 hemisection (C2Hx) injury on day 0 and then on a weekly basis afterwards either for 1 ( A ) or 4 weeks ( B ). For the terminal 1-week post-injury timepoint ( A ), subjects also were treated with intermittent hypoxia treatment and evaluated for ventilatory long-term facilitation using whole-body plethysmography. At their experimental endpoint, all subjects were subjected to terminal diaphragm electromyography prior to euthanasia while under anesthesia. For the 1-week timepoint ( A ), subjects’ cervical spinal cords were collected. Panel C depicts the pre-injury and weekly post-injury baseline and gas challenge paradigm to which all subjects from both the 1-week and 4-week post-injury timepoints were exposed. This included acclimation time prior to a baseline recording, after which subjects were exposed to a moderate hypoxia challenge, followed by a recovery period. Subsequently, subjects were exposed to a more severe hypoxic-hypercapnic challenge and then completed the protocol with recovery on room air. Panel D depicts the intermittent hypoxia (IH) treatment imposed upon subjects within the 1-week post-injury timepoint wherein ventilatory outputs were measured at baseline, during IH, and for 1 hour after the end of IH to measure long-term facilitation. After warming and gently mixing the solution, clodronate liposomes in pH-balanced PBS (Clophosome ® -A, Cat# F70101C-A, FormuMax, Sunnyvale, CA, USA) were injected into the tail vein of isoflurane-anesthetized animals on days 1, 3, and 6 post-injury ( Figure 2A – B ) 6 , 9 , 11 . Subjects assigned to control underwent the same procedure, but with 2 mL of physiologic saline as appropriate per previously published procedure 26 , 27 . One subject displayed a short period of apnea and irregular respiration lasting approximately 5 minutes during recovery from anesthesia for tail vein injection while some injections seemed to extravasate into subcutaneous, rather than intravenous compartments. This affected a portion of 30/78 doses across 26 subjects, equally split between clodronate and saline groups and timepoints ( Figure S1C ). 2.4. Ventilatory measurement All ventilatory measurement was conducted following acclimation via a small animal whole-body plethysmography (WBP) system (DSI, Buxco ® , FinePointe) using a 3 L/min flow rate, measured both at baseline and in response to both a hypoxic and hypoxic-hypercapnic challenge pre- injury and on a weekly basis post-C2Hx until endpoint ( Figure 2A – C ). Some tidal volume and minute ventilatory data which was inadvertently collected using incorrect parameters were excluded. Sample sizes included for each outcome measure and treatment group are described in Table 1 . Table 1. Distribution of subjects per outcome measure by treatment Post-Injury Weight Change −Clodronate +Clodronate −Carprofen 21 20 +Carprofen 23 22 Diaphragm Electromyography 1-week post-injury cohort 4-week post-injury cohort −Clodronate +Clodronate −Clodronate +Clodronate −Carprofen 3 4 15 15 +Carprofen 5 3 15 14 Respiratory Rate (WBP) −Clodronate +Clodronate −Carprofen 19 20 +Carprofen 20 18 18 Tidal Volume and Minute Ventilation (WBP) −Clodronate +Clodronate −Carprofen 14 15 +Carprofen 16 15 11 IH and Long-Term Facilitation (WBP) 1-week post-injury cohort −Clodronate +Clodronate −Carprofen 3 5 +Carprofen 5 4 CatWalk −Clodronate +Clodronate −Carprofen 11 14 +Carprofen 15 18 Open in a new tab Additionally, subjects’ ventilatory output in the terminal 1-week post-injury timepoint was evaluated via WBP before, during, and after exposure to intermittent hypoxia treatment (IH) ( Figure 2D ), similar to previously published procedure from our lab to assess breathing plasticity known as long-term facilitation (LTF). 2.5. Locomotor assessment Locomotor assessments were made using the CatWalk XT Gait Analysis system (Noldus Information Technology b.v., Wageningen, The Netherlands) after acclimation according to the timeline depicted in Figure 2A – B . A single researcher conducted assessments in a dark room, using 11.0 dB camera gain, 0.10 intensity threshold, 17.5 V red ceiling light, 15.0 V green walkway light, and a minimum green intensity value of 85 with the video camera adjusted to 29 inches beneath the walkway with fully open aperture. Runs of duration outside of the 0.5–12 second range and/or those with maximal variation exceeding 90% were deemed non-compliant and excluded. Three compliant runs were required for successful trial completion on each day, and subjects’ data were excluded from that timepoint if they failed to complete at least 1 compliant run within 15 minutes of first walkway placement. After automatic paw-print classification was performed using the CatWalk software, remaining unidentified signals were manually reviewed by a researcher unblinded to treatment group. 2.6. Terminal diaphragm electromyograph recordings Upon reaching the targeted timepoint of either 1-week (8–11 DPI) or 4-weeks (28–30 DPI), terminal diaphragm electromyograph (EMG) recordings were performed for all subjects while under isoflurane anesthesia after anterior surgical exposure of the trachea. Similar to previously published procedure from our laboratory, diaEMG recordings were conducted via laparotomy and insertion of pairs of Chalgren unipolar needle electrodes (CWE, Inc., Gilroy, CA) into the dorsolateral quadrant of each costal hemidiaphragm with appropriate grounding 22 . A Power1401–3 data acquisition system and Spike2 8.08 software (CED, Cambridge, UK) along with a BMA-400 AC/DC Bioamplifier (CWE, Inc., Ardmore, PA) were used to acquire and interpret the electromyographical signal 22 . A10-minute baseline recording was completed prior to tracheal occlusion using hemostats to provoke maximally increased respiratory drive. Following tracheal occlusion-induced apnea, the recording was terminated and the animals were euthanized by exsanguination while under terminal isoflurane anesthesia. Data from 3 subjects’ EMG recordings were excluded from final analysis due to confounding factors present during the procedure. 2.7. Data analysis 2.7.1. Ventilatory data As in previously published work, WBP data from subjects’ weekly baseline/gas challenge and IH measurements was exported from FinePointe software and MATLAB ® (MathWorks ® , version R2022a) scripts were utilized to calculate tidal volume (V T ), respiratory rate (RR), and minute ventilation (V E ) and parse data according to treatment type, timepoint, and targeted outcome 28 . We normalized all of the current study’s V T and V E data to each subject’s body mass prior to injury. For our weekly ventilatory data, we report average V T , RR, and V E during the final 10 minutes of normoxic baseline and during both 10-minute-long periods of hypoxia and hypoxia-hypercapnia. Additionally, we express the average of the final 10 minutes of baseline as a percent of the average V T , RR, and V E expressed during hypoxic hypercapnia as a proxy indicator of baseline respiratory drive relative to maximal effort. As previously published, outcomes for IH data analysis included subjects’ ventilatory output during hypoxic exposure and ventilatory long-term facilitation (LTF) for 1-hour following the end of IH 28 . For each subject’s response to hypoxia, we calculated the ratio of each subject’s ventilatory output (V T , RR, and V E ) during the 5 hypoxic exposures to that of the immediately preceding normoxic condition, expressing the result as a percent. For LTF, we compared the average of the final 10 minutes of pre-IH baseline and each minute of the hour-long post-treatment recording, expressing each ventilatory metric as a percent ratio of post-IH to pre-IH output with respect to V T , RR, and V E between treatments and across time. A value significantly greater than the pre-IH timepoint (100%) was interpreted to indicate LTF. 2.7.2. Locomotor data We excluded CatWalk variables relying on interactive footprint measurements, and selected 17 other variables for analysis based on their qualitative relevance to the C2Hx injury model and a survey of the CatWalk/SCI literature 29 – 45 . These variables assessed general and limb-specific locomotor function across all four limbs, favoring aggregate measures over individual components 29 – 45 . Secondarily, we used the Classification Learning Automated Software System (CLASSify, University of Kentucky Center for Applied Artificial Intelligence) to assess whether other, less intuitive CatWalk variables might better capture C2Hx-driven locomotor differences, using the full multi-study dataset previously referenced 46 . Results did not indicate any necessary changes to the originally determined set of 17 variables. 2.7.3. Electromyography data EMG data files were anonymized for blinded assessment and Spike2 8.08 software (CED, Cambridge, UK) was used to assess average integrated hemidiaphragm inspiratory amplitude ipsilateral to injury during two 30 second baseline epochs and one 6-second epoch containing the highest amplitude inspiratory activity induced by terminal tracheal occlusion. These values were used to calculate the baseline percentage of maximal inspiratory output as described in previous publication 22 . We categorically analyzed the number of subjects with and without ipsilesional inspiratory hemidiaphragm activity by treatment group in addition to quantitative analyses comparing differential diaphragm activity and diaphragm recovery by treatment. Analyses of diaphragm activity included all subjects, but analysis of diaphragm recovery excluded data from subjects with a baseline percentage of maximal output equal to 0. 2.8. Statistical analysis Data from the 1 and 4-week post-injury cohorts is graphed and analyzed via controlled longitudinal statistical models as a single pooled cohort within each treatment group for the outcomes of weight, CatWalk, and weekly ventilatory measurements. Terminal diaphragm EMG is analyzed separately at 1 and 4WPI since experiments were not powered to carry out longitudinal comparisons of diaEMG by timepoint. Ventilatory plasticity was only evaluated within the 1WPI cohort and is analyzed alone. Primary analyses were performed using longitudinal mixed models to account for repeated measures. Models included a within-subjects factor of time, between-subjects’ factors of clodronate and carprofen exposure, and all higher-order interactions for the outcomes of post-injury weight-change, weekly ventilatory output, ventilatory plasticity following IH treatment, and locomotor recovery. CatWalk locomotor variables of regularity index and the proxy measure of each forepaw’s maximal weight-bearing ability (Max Contact Max Intensity) were non-normal and underwent a Box-Cox transformation prior to analysis. Significant interactions were followed up post-hoc pair-wise comparisons to determine the locus of the significant effect. The tenability of model assumptions was verified for all dependent variables. Because baseline values used in post-injury percent weight change and in ventilatory plasticity analyses were tautologically defined as 100%, analyses using previously described longitudinal mixed models were also conducted to evaluate whether each group’s mean value at a given timepoint significantly differed from baseline. Non-longitudinal ANOVAs of pre-injury weights and total supplemental saline were pooled between 1WPI and 4WPI cohorts, as were all categorical analyses of CatWalk locomotor failure, differential attrition, and injection extravasation across treatments. Pre-injury, pre-intervention weight data and total saline administration was analyzed by separate Kruskal-Wallis one-way ANOVAs on ranks to compare body weight and saline administration across our four treatment groups. Importantly, graphed data remains unranked, simply so that the numerical weight values remain meaningful to readers. Though 4WPI output and recovery diaEMG data did not meet normality assumptions for 2-way ANOVA, rank transformation did not result in substantial normalization. As a result, reported results are from the non-ranked parametric test. All diaEMG data from 1WPI and ventilatory output during IH treatment passed both normality and homogeneity of variance testing and thus were not ranked for analysis. Categorical analyses of the dichotomous outcomes of EMG recovery, locomotor trial failure, overall attrition, and suspected tail vein extravasation, were conducted via Chi-squared tests. For locomotor trial failure, a Chi-squared test with pairwise post-hoc comparisons to the saline-treated control group was performed with adjustment for multiple comparisons to account for the absence of failures in the saline + carprofen group. The incidence of suspected extravasation was analyzed across treatment groups as a single four-level independent variable using a Chi-squared test to determine whether rates of extravasation differed between groups as a complication of injection, without separately evaluating the effects of clodronate or carprofen. To address whether potential clodronate extravasations affected recovery, data from all clodronate-treated subjects was pooled and regressions were run to examine the relationship between the number of injections (0, 1, or 2) with extravasation and each quantitative, non-categorical outcome variable. All trend lines are graphed with corresponding R 2 and p-values. For all analyses, a p < 0.05 was utilized for significance. All p-values are reported within the results with no corrections for multiple comparisons. All graphs were generated with GraphPad Prism (GraphPad Software, Boston, MA) and data analysis was performed using SAS v 9.4 (SAS Institute INC, Cary, NC), SigmaPlot (Systat Software INC, San Jose, CA), GraphPad Prism, and Python with the SciPy library. Python coding was done via ChatGPT only for post-hoc and effect size testing subsequent to Chi-square analysis after non-ideal model performance on binomial logistic regression for analysis of locomotor trial failure. Conceptual figures were created using Microsoft PowerPoint ® . 3. Results 3.1. Pre-injury weight significantly differed by group prior to any differential treatment intervention Weights from all subjects with attrition attributable to drug exposure were included in all weight analyses. A Kruskal-Wallis one-way analysis of variance on ranks was performed to compare pre-injury pre-treatment body weight across our four treatment groups due to non-normality by Shapiro-Wilk test, though non-ranked weights are used in Figure 3A for greater interpretability. Results demonstrated a statistically significant difference in median weights among the groups prior to any experimental manipulation with post-hoc pairwise comparisons (Dunn’s method) indicating that the control group had lower body weight than each other group. Figure 3. Pre-injury weight significantly differed prior to any differential treatment intervention and clodronate and carprofen significantly worsened post-injury weight-change. Open in a new tab Subjects assigned to the saline-treated group had lower absolute body weight prior to injury and any treatment (Panel A) . Because of violations of normality, a Kruskal-Wallis one-way ANOVA on ranks was used to compare pre-injury weights, with significant pairwise comparisons indicated by ** (p < 0.01) (Panel A). When body weight was expressed as percent change from each subject’s pre-injury value (Panel B), clodronate and carprofen-treated groups experienced greater and more acute post-injury weight loss and more delayed recovery. A longitudinal mixed model assessed the effects of clodronate, carprofen, and their interaction across time in addition to each treatment group’s difference from baseline (100%) at each timepoint. For Panel B , *p < 0.05, **p < 0.001, and ***p < 0.0001, with colored asterisks reflecting the timepoints at which corresponding treatment groups exhibited significant deviations from baseline. Black asterisks indicate that the deviation was significant across all groups. Significant pairwise comparisons are indicated for timepoints by vertically ordered colored shapes corresponding to each treatment group. Vertical lines indicate specific pairwise comparisons indicated as significant by a p-value < 0.05 and ^ symbol. 3.2. Clodronate and carprofen treatments significantly worsened post-C2Hx weight change Data from all subjects with attrition attributable to drug exposure was included in analysis of longitudinal post-C2Hx weight change and demonstrated significant two-way and 3-way interactions of clodronate*time ( Figure 3B ). Additionally, clodronate and clodronate + carprofen-treated groups were the first to significantly lose weight at 2 days post-injury while all treatment groups had significant weight loss from day 3 through week 1 post-injury ( Figure 3B ). The saline-treated group was the first to recover weight to pre-injury levels while all other groups’ weight remained significantly attenuated at 2WPI. All three clodronate and/or carprofen-treated groups returned to baseline weight by 3WPI, while the saline-treated group weight was actually increased at that timepoint. 4WPI weight was unchanged from baseline in any of the four treatment groups. Significant pairwise comparisons at each timepoint are depicted in Figure 3B . 3.3. Carprofen treatment significantly improved recovery of inspiratory diaphragm activity at 1, but not 4-weeks post-C2Hx Examples of diaEMG traces from subjects with and without fictive breathing activity ipsilateral to C2Hx at both 1WPI and 4WPI are depicted in Figure 4Ai and Bi , respectively. A binary logistic regression was performed to assess whether clodronate treatment, carprofen treatment, or their interaction predicted activity. Neither at 1 nor at 4WPI did clodronate, carprofen, or their interaction attain statistical significance ( Figure 4Aii & 4Bii ). At 1WPI, neither clodronate, carprofen, nor their interaction significantly affect the amplitude of baseline inspiratory diaphragm output by 2-way ANOVA ( Figure 4Aiii ). Interestingly, carprofen did significantly improve diaphragm recovery when diaphragm output was analyzed without inclusion of subjects exhibiting an absence of baseline activity, but no clodronate main effect or clodronate*carprofen interaction attained significance ( Figure 4Aiv ). At 4WPI, neither clodronate, carprofen, nor their interaction significantly affected diaphragm output or recovery by 2-way ANOVA ( Figure 4Biii – 4Biv ). Figure 4. Carprofen treatment may significantly improve recovery of inspiratory diaphragm activity by the 1-week post-C2Hx timepoint, but no significant treatment-induced differences are present at 4-weeks post-injury. Open in a new tab Columns A and B depict diaphragm electromyography data ipsilateral to C2Hx in animals recorded at the 1-week post-injury and 4-weeks post-injury timepoints, respectively. Row i contains an example of an diaphragm electromyogram raw trace from an animal without fictive breathing activity (left) and a trace from an animal with fictive breathing (right) for each post-injury timepoint. Sample EMG traces taken from subjects near quantitative mean among saline-treated subjects. Row ii contains categorical analysis of subjects with and without fictive breathing activity by treatment, analyzed by binary logistic regression. Rows iii and iv depict quantitatively analyzed EMG data, expressed as baseline percent of maximal amplitude, depicted as mean ± SEM, and tested by 2-way ANOVA. Row iii contains analysis of all data, including those animals which exhibited values of zero indicating no baseline inspiratory activity. Row iv depicts data with all zero values excluded, thus only comparing the quantitative degree of recovery between treatment groups at both timepoints 3.4. Clodronate and carprofen synergistically impaired ventilatory recovery post-C2Hx Representative ventilatory waveform traces recorded via WBP are depicted in Figure 5A . C2Hx injury significantly affected ventilatory output across all metrics except for minute ventilation at baseline ( Figure 5Di ) as evidenced by a significant main effect of time. Clodronate and carprofen synergistically diminished minute ventilation at baseline ( Figure 5Di ) and during hypoxia ( Figure 5Dii ) specifically at 3 and 4WPI by post-hoc pair-wise comparison. Furthermore, clodronate and carprofen significantly increased baseline breathing effort relative to output during hypoxic-hypercapnic challenge with significant pairwise differences present from 1–4WPI ( Figure 5Civ ). Figure 5. Clodronate and carprofen synergistically impair ventilatory recovery across 4-weeks post-injury. Open in a new tab Panel A depicts sample ventilatory waveforms recorded by WBP of a subject belonging to the saline treatment group prior to C2Hx (top) and 1-week post-injury (bottom) during the final 5 minutes of baseline, hypoxic challenge, and hypoxic-hypercapnic challenge (left to right, respectively.) Overall, C2Hx injury significantly affected ventilatory output across all metrics and conditions except for minute ventilation at baseline ( Panel Di ) as evidenced by a significant main effect of time. Clodronate and carprofen synergistically diminish minute ventilation at baseline ( Panel Di ) and both drug treatments impair recovery of minute ventilation during hypoxic exposure ( Panel Dii ). Furthermore, clodronate and carprofen lead to differential baseline respiratory rate output when expressed as percent of output during hypoxic-hypercapnic challenge ( Panel Civ ). Significant pairwise comparisons are indicated between treatment groups at specific timepoints for Panels Di-ii and Civ where justified by significant 3-way interaction. In all panels, the −1 timepoint on the x-axis depicts pre-injury. Statistics reported for each panel include the main effect of time as well as 2-way and 3-way interactions of clodronate*time, carprofen*time, and clodronate*carprofen*time, indicated by longitudinal mixed model, with p-values < 0.05 bolded where appropriate. Pairwise comparisons marked with ^ and a vertical line indicate p-value < 0.05. 3.5. Clodronate and carprofen-exposed groups exhibited less frequent and shorter episodes of increased ventilation post-IH treatment than saline-treated subjects 1WPI Figure 6A depicts representative WBP ventilatory waveforms for two saline and clodronate + carprofen-treated subjects before, during, and following IH treatment. To assess LTF expression, we examined time periods equal to or greater than 3 minutes in which treatment groups expressed ventilatory output that significantly deviated from baseline (100%) over the 1-hour post-IH timeframe. The saline-treated group demonstrated 5 periods of significant facilitation of V T , RR, and V E while clodronate-treated subjects exhibited 1 period of V T depression and 2 of RR and V E facilitation ( Figure 6Ci – iii ). No significant change was present within either saline + carprofen or clodronate + carprofen-treated groups ( Figure 6Ci – iii ). Figure 6. Saline-treated control subjects exhibit more frequent episodes of prolonged ventilatory increase after intermittent hypoxia treatment than clodronate or carprofen-exposed groups at 1-week post-C2Hx. Open in a new tab Panel A shows representative whole-body plethysmography (WBP) waveforms from a subject in the saline-treated group (top) and the clodronate + carprofen group (bottom), recorded during the final 5 minutes prior to IH, the first hypoxic bout, and 30 minutes post-IH (left to right, respectively). Column B summarizes ventilatory output during ongoing IH exposure; no significant effects of clodronate or carprofen were detected. Panels Ci and Cii show tidal volume and respiratory rate during the 1-hour post-IH period; again, no significant main effects or interactions were observed in the factorial model. Statistical results in Column B reflect p-values from two-way ANOVAs. In Column C , p-values are derived from two- and three-way interactions in a longitudinal mixed model evaluating the effects of clodronate, carprofen, and time. Significant p-values (p < 0.05) would be bolded if present. A complementary group-based model was used to test each treatment group’s deviation from baseline (100%) at each post-IH timepoint in Column Ci-iii . Colored asterisks and accompanying bars indicate time intervals where a given treatment group showed a significant deviation from baseline (p < 0.05). To reduce spurious findings, only periods with at least three consecutive significant timepoints are shown. Notably, neither the saline + carprofen nor clodronate + carprofen groups exhibited a sustained (≥3-minute) deviation from 100% baseline in any ventilatory measure during the 1-hour post-IH period (Panels Ci–Ciii) . Statistical analysis of the main effects and interactions of clodronate and carprofen across time indicated that neither drug nor their interaction significantly enhanced or depressed hypoxic ventilatory output or LTF over the 1-hour post-IH recording period for V T , RR, or V E ( Figure 6Bi – iii & 6Ci – iii ). 3.6. Clodronate treatment increased right hindpaw compensation for injury-induced motor deficits following C2Hx, but carprofen did not significantly affect locomotor recovery Because of the poor fitting of our originally planned binary logistic regression, a Chi-Square test was used to test between assess whether clodronate treatment, carprofen treatment, or their interaction predicted locomotor failure. The overall chi-square test was significant, indicating a potential association between treatment groups and locomotor trial failure. Post-hoc pairwise chi-square tests, with Bonferroni correction, were conducted to further investigate differences between the groups, but indicated no significant differences between groups ( Figure 7A ). Figure 7. Preliminary statistical analyses, analysis parameters, and visual depiction of CatWalk testing following C2Hx injury. Open in a new tab Panel A demonstrates no pairwise difference in the numbers of subjects per treatment group with failure to complete a compliant run of CatWalk at 1 or 2 weeks post-injury, as compared to saline-treated control, indicated by results of an omnibus chi-square test and post-hoc pairwise testing. As justified by non-significant 3-way interaction of clodronate, carprofen, and time according to our longitudinal mixed model, the two-way comparisons of clodronate with time and carprofen with time were graphically depicted independently from one another in the following two figures. Panel B demonstrates the makeup of those pairs of groups as collapsed across clodronate and carprofen independent variables. Panel C exemplifies a sample of the prints identified during one of CatWalk for one animal belonging to the Saline treatment group at pre-injury (top) and 1-week post-injury timepoints (bottom). Notice the less coordinated footfall pattern and diminished contact areas of particularly left forepaw (LF) and hindpaw (LH) placement due to C2Hx. Analysis of locomotor recovery revealed no significant three-way interactions of clodronate*carprofen*time for any dependent variable within that datasets ( Table 2 ). Thus, data was collapsed across individual treatment groups as described in Figure 7B to more clearly depict the separate interactions of clodronate*time and carprofen*time in separate figures. Figure 7C depicts paw prints detected and measured via CatWalk for a single subject within the saline-treatment group at pre-injury (top) and 1WPI timepoints (bottom). Table 2. Summary of non-significant 3-way interactions indicated by longitudinal mixed model analysis of CatWalk locomotor recovery Outcome Clodronate*carprofen*time p-value Number of Steps 0.2505 Regularity Index 0.4567 Mean Speed 0.3531 LF Print Area 0.4128 LF Maximal Weight-Bearing 0.7883 LF Attributable Speed 0.3721 LH Print Area 0.9840 LH Maximal Weight-Bearing 0.8847 LH Attributable Speed 0.0512 RF Print Area 0.6137 RF Maximal Weight-Bearing 0.4105 RF Attributable Speed 0.2120 RH Print Area 0.8235 RH Maximal Weight-Bearing 0.9146 RH Attributable Speed 0.1259 Open in a new tab Figure 8Av depicts the only statistically significant interaction of clodronate*time for locomotor output, demonstrating that clodronate treatment increases right hindpaw print area at 2WPI and 4WPI compared to control. No statistically significant interaction of carprofen*time was identified by our longitudinal mixed model for any selected CatWalk metric ( Figure 9 ). Figure 8. Clodronate treatment significantly increases right hindpaw compensation for injury-induced motor deficits. Open in a new tab Across all locomotor metrics, a significant main effect of time for all panels indicates that C2Hx significantly affects locomotor function as measured by CatWalk. Overall locomotor metrics are within the first row ( i ) while limb-specific parameters are depicted in order from left forelimb, left hindlimb, right forelimb, and right hindlimb in rows ii through v respectively. The “regularity index” (Panel Bi ) indicates interlimb coordination. In rows ii-v , column A depicts print area for each paw, column B contains the variable of “maximum contact maximum intensity” for each paw, and column C contains measurements of “body speed” for each limb. Print area is a metric describing the surface area of each paw contacting the platform during locomotion, as an indication of functional paw use, seen in rows ii-v, column A . Maximum contact maximum intensity is a proxy measure for maximal weight-bearing ability for each paw depicted in rows ii-v, column B . Finally, “body speed” is a measure of the component of overall walking speed attributable to each limb, rows ii-v, column C . In all panels, the −1 timepoint on the x-axis depicts pre-injury. The clodronate group compared in this figure is collapsed across the individual clodronate and clodronate + carprofen-treated groups described in previous analyses, with saline comprised of saline and saline + carprofen groups. This collapsed group comparison approach was justified by the absence of statistically significant 3-way interactions of clodronate, carprofen, and time according to our longitudinal mixed model. Thus, the statistical results reported on each panel are the p-values only associated with the clodronate*time interaction. P-values < 0.05 are bolded, while * indicates individual timepoints at which the clodronate versus saline comparison is significant by post-hoc test. Figure 9. Carprofen treatment does not significantly affect locomotor recovery during the 4-week post-injury time period. Open in a new tab Across all locomotor metrics, a significant main effect of time for all panels indicates that C2Hx significantly affects locomotor function as measured by CatWalk. Overall locomotor metrics are within the first row ( i ) while limb-specific parameters are depicted in order from left forelimb, left hindlimb, right forelimb, and right hindlimb in rows ii through v respectively. The “regularity index” (Panel Bi ) indicates interlimb coordination. In rows ii-v , column A depicts print area for each paw, column B contains the variable of “maximum contact maximum intensity” for each paw, and column C contains measurements of “body speed” for each limb. Print area is a metric describing the surface area of each paw contacting the platform during locomotion, as an indication of functional paw use, seen in rows ii-v, column A . Maximum contact maximum intensity is a proxy measure for maximal weight-bearing ability for each paw depicted in rows ii-v, column B . Finally, “body speed” is a measure of the component of overall walking speed attributable to each limb, rows ii-v, column C . In all panels, the −1 timepoint on the x-axis depicts pre-injury. The carprofen group compared in this figure is collapsed across the individual saline+carprofen and clodronate + carprofen-treated groups described in previous analyses, with control comprised of saline and clodronate-treated groups. This collapsed group comparison approach was justified by the absence of statistically significant 3-way interactions of clodronate, carprofen, and time according to our longitudinal mixed model. Thus, the statistical results reported on each panel are the p-values only associated with the carprofen*time interaction. P-values < 0.05 would be bolded if present. 3.7. Neither attrition rate, total saline administration, nor tail vein injection extravasation significantly differed by treatment group We performed a multiple logistic regression to assess whether clodronate treatment, carprofen treatment, or their interaction predicted attrition, but found no statistically significant association ( Figure S1A ). Additionally, we found no significant difference between total post-surgical administration of saline between groups, utilizing a one-way Kruskal-Wallis ANOVA on ranks because of non-normal data ( Figure S1B ). Finally, we assessed the relationship between treatment groups and the occurrence of possible tail vein injection extravasation using a Chi-square test, finding no significant association between the treatment groups and extravasation failure ( Figure S1C ). Observationally, subjects affected by the apparent extravasation suffered no observable discomfort upon recovering from anesthesia and their tails regained normal pliancy within hours. 3.8. Clodronate tail vein extravasation was not associated with weight change or ventilatory recovery, but was inconsistently associated with metrics of diaphragm output, breathing plasticity, and locomotion post-C2Hx Within all clodronate-treated subjects (collapsed across carprofen exposure), we performed linear regressions for post-injury weight change, diaEMG, ventilatory output and plasticity, and locomotor recovery within each timepoint (or 15-minute bin, for ventilatory plasticity) with respect to each subject’s noted number of injections with possible extravasation (0, 1 or 2). This was to ascertain whether the degree of functional output due to clodronate might correlate with the possible extravasation observed for each subject. Results indicate that clodronate extravasation was not significantly associated with either post-injury weight change ( Figure S2 ) or weekly ventilatory output as measured by WBP ( Figure S4 ). However, diaphragm output at 1WPI was identified to be significantly related to extravasation ( Figure S3Ai ), as was respiratory rate and minute ventilation LTF during the 1 st and 3 rd 15-minute bins of the hour-long post-IH recording ( Figure S5Bii – Biii ) in addition to the CatWalk parameters of both left and right hindpaw print area at 2WPI ( Figure S6Aiii & Av ), and left hindpaw maximal weight-bearing at 2WPI ( Figure S6Biii ). A more complete reporting of those statistically significant results can be found in Table S1 . 4. Discussion In this study we examined the hypothesis that both liposomal clodronate and carprofen treatment would improve breathing and locomotor recovery acutely following the C2-level hemisection model of experimental SCI in rats. Our data indicates that liposomal clodronate treatment does improve locomotor function after experimental SCI as hypothesized, but along with carprofen acts to paradoxically impair ventilatory function. Because our investigation focused on functional breathing and locomotor outcome, we did not include histological analyses which would define the underlying cellular and molecular mechanisms. Consequently, future work will be vital to determine whether clodronate-mediated macrophage depletion or carprofen-induced COX-2 inhibition within the injured spinal cord or peripheral tissues accounts for these effects. Nevertheless, the following discussion integrates our results with relevant literature to outline plausible mechanisms, highlight directions for future mechanistic investigation, and discuss potential confounds. Prior studies examining COX-2 inhibition and clodronate-mediated macrophage depletion provide a framework for interpreting the present results. Inhibition of COX-2, the preferential target of carprofen, can be neuroprotective after SCI, attenuating maladaptive inflammation and oxidative stress to ameliorate motor deficits 17 – 21 , 24 , 25 , 47 – 81 . Furthermore, clodronate-mediated depletion of MDMs can diminish maladaptive inflammatory processes and lead to tissue sparing, diminished axonal retraction, and improved functional locomotor recovery following experimentally induced T8–9 contusion, C8 dorsal column crush, and ischemic models of SCI 6 , 8 – 14 . Studies of clodronate after lateral hemisection at the T8 spinal level also demonstrate spinal cord macrophage depletion and significant locomotor improvement, as well as attenuation of lesion volume, cavitation, and axonal die-back 7 . However, these results are difficult to interpret with respect to clodronate’s efficacy alone because of Grosso et al.’s exclusive evaluation of clodronate in combination with other therapies like rolipram and chondroitinase ABC 7 . In our study, we utilized the C2Hx model of SCI to induce breathing deficits well suited for novel study of clodronate’s efficacy as a breathing therapeutic. In contrast to the C2Hx model, contusion injuries lead to less consistently severe breathing deficits even when applied at high cervical levels 82 – 85 . However, since the C2Hx model likely has a signature of secondary inflammation and damage distinct from that initiated by non-laceration models of SCI utilized in prior study of clodronate (and COX-2 modulators like carprofen), it is worthwhile to discuss how the breathing and locomotor outcomes in the current study were impaired rather than improved by clodronate and carprofen treatments. Contusions lead to widespread disruption of the blood-spinal cord barrier 86 – 88 . In contrast, a C2Hx likely induces a more localized blood-spinal cord barrier disruption than a contusion, though the extent of primary hemorrhage and secondary blood-spinal cord barrier disruption has not been as exhaustively investigated after C2Hx and has not been directly compared with that induced by contusion SCI models 88 – 91 . As a result, the magnitude of secondary inflammation post-C2Hx, as well as the extent to which hematogenous monocytes infiltrate the damaged spinal cord from the peripheral vascular system might be more limited than in contusion. To investigate this, future immunohistochemical study would be insightful to measure the comparative magnitude of CD68+ or Iba1+ cells within the epicenter and penumbra of both C2-hemisected and cervically contused animals, both in the absence and presence of clodronate treatment. Co-staining for COX-1 and COX-2 would be also prove valuable to understand the extent of the potentially neuroprotective immunomodulatory effects of carprofen in C2Hx or contusion. In addition to the centrally-focused immunomodulatory mechanisms which might mechanize our results, it is necessary to discuss our treatments’ effects on the respiratory system. Intravenous administration of clodronate liposomes has been shown to deplete pulmonary intravascular macrophages as well as circulating monocyte populations and attenuate metrics of inflammation and histological damage in experimental models of acute lung injury (ALI) 92 – 97 . Interestingly, these results parallel the field of SCI research which inspired the current project, namely that MDMs may play a more pathological role than resident macrophages in the immune response to localized pathology 6 , 9 , 11 , 12 , 92 – 97 . Depletion of alveolar macrophages via systemic or airway administration of liposomal clodronate can lead to increased respiratory rate, more profound cytokine/chemokine release, greater cell-mediated lung inflammation, and exacerbated histological damage in lung injury 27 , 94 , 98 – 104 . However, attenuation of the alveolar macrophage population can be beneficial in experimental models of obesity-induced, ventilator-induced, and radiation-induced lung injury, particularly with respect to alveolar barrier permeability 105 – 108 . As a key connection between the lung and SCI, C2Hx causes metabolic disruptions within the lung in addition to various metrics of inflammation as measured via cell and cytokine/chemokine analysis of bronchoalveolar lavage fluid (BALF) which point to a phenotype consistent with mild ALI/acute respiratory distress syndrome (ALI/ARDS) 23 . C2Hx also induces lung edema, indicative of increased vascular/alveolar permeability which is a hallmark of ALI/ARDS 23 . Indeed, more caudal models of SCI also induce lung edema, hemorrhage, and inflammation 109 , 110 . Furthermore, a model of bleomycin-induced ALI in the murine animal causes brainstem inflammation via peripheral-to-central immune communication specifically via peripheral cytokine signaling and cyclooxygenase (COX) induction at brainstem structures in proximity to areas such as the nucleus tractus solitatarius (NTS) which is the primary peripheral chemoreceptor afferent integration center in the neural control of breathing 111 – 114 . Ultimately, this sterile ALI induced increased respiratory rate and diminished biologically adaptive breathing patterns 113 , 114 . Comprehensively with respect to ventilatory function, the results of our current study combined with findings from these various fields suggest that even systemically administered liposomal clodronate following C2Hx might exploitatively extravasate into the alveolar airspace due to SCI-induced lung/vascular barrier permeability and deplete populations of alveolar macrophages. As a result, intravenous administration of liposomal clodronate might exacerbate the development of ALI following C2Hx, leading to peripheral-to-central trafficking of inflammatory mediators to brainstem respiratory centers and the worsened recovery of ventilatory function identified in the current study. Our findings that clodronate interacted significantly to induce an increase in baseline respiratory rate relative to hypoxic-hypercapnic challenge ( Figure 5Civ ) particularly suggest an ALI-like phenotype following C2Hx 98 , 113 , 114 . Future studies are needed to determine whether this mechanism truly underlies clodronate’s ventilatory effects after C2Hx. Such work should include immunohistochemical assessment of brainstem inflammation, histological analysis of lung tissue for potential exacerbated pathology, and bronchoalveolar lavage assays to evaluate macrophage depletion and inflammatory cell or cytokine profiles. In explanation of the potential mechanism whereby carprofen contributed to the impairment of ventilatory recovery, we focus upon cyclooxygenase and its impact upon breathing and lung function. COX-1 is preferentially expressed in the airway and can serve an immunoregulatory function, diminishing inflammatory activation such as occurs in asthma, while the inducible COX-2 enzyme tends to exacerbate diseases of airway inflammation 115 . Paradoxically, COX-2 and its downstream products have also been shown to serve an airway-protective role, but COX-2 inhibition can also attenuate airway hyperresponsiveness and improve lung pathologies such as ventilator-induced lung injury and fibrotic disease 116 – 121 . COX-2 expression following some lung injuries seems to be localized to populations of alveolar macrophages 119 . Following C2Hx, we suggest that carprofen attenuates ventilatory recovery via maladaptive COX-1 and COX-2 inhibition in the lung which exacerbates SCI-related lung injury and inflammation, affecting respiratory centers within the brainstem and spinal cord via peripheral-to-central pathways previously described 113 , 114 . To investigate this, future studies should first investigate whether selective COX-1 or COX-2 inhibition following C2Hx differentially affect lung pathology and ventilatory recovery. Subsequently, eicosanoids downstream of COX-1 and COX-2 could be targeted to provide better mechanistic specificity. The results of our examination of diaphragm electromyography and ventilatory plasticity following C2Hx, clodronate, and carprofen treatment lend further insight into breathing outcome. Though carprofen treatment significantly increased diaphragm recovery ipsilateral to injury at 1WPI, no such results were present at the 4WPI timepoint ( Figure 4Aiv & Biv ). These results suggest an acutely neuroprotective effect of carprofen with respect to preservation of bulbospinal motor innervation to the phrenic motor pool ipsilateral to C2Hx injury, consistent with reports on the effects of COX-2 inhibition after other models of experimental SCI 17 – 21 , 24 , 25 , 47 – 81 . The acute and limited nature of this effect is hardly surprising, given that carprofen administration in our experiment ended on the second post-injury day. However, ventilatory recovery at 1WPI was not improved by carprofen treatment ( Figure 5 ). Given this, along with the consideration of this analysis’ low statistical power, our findings suggest that the single significant EMG result should not be attributed substantial functional benefit or importance. This study’s moderate IH treatment is known to induce ventilatory plasticity known as LTF which depends upon serotonergic signaling within the phrenic motor nucleus 122 – 127 . LTF is a form of plasticity capable of restoring functional breathing outputs following SCI, but usually cannot be induced until 4 or even 8-weeks post injury, due to acute loss of serotonergic innervation and cross-talk inhibition by inflammatory-mediated activation of a competing adenosine-dependent pathway to LTF 123 , 128 – 134 . We initially hypothesized that clodronate and carprofen would remove the inflammatory brake and enhance expression of ventilatory LTF even at 1WPI. However, comparison of each treatment group’s change in ventilatory output during the 1-hour post-IH recording period demonstrates that clodronate and carprofen-exposed subjects less frequently express ventilatory LTF than saline control ( Figure 6Ci – iii ). Non-significant trends also indicate that the two drugs’ combination might synergistically depress minute ventilation after IH, rather than facilitate it. In context of the field of research concerning IH and LTF, these results suggest that clodronate and carprofen treatment could diminish adenosinergic substrate availability and thus attenuate breathing plasticity at the acute 1WPI post-C2Hx timepoint 122 – 134 . However, future experiments utilizing selective 5-HT 2A and adenosine antagonists along with clodronate and carprofen treatments during IH treatment are necessary to test this proposed mechanism. With respect to locomotor recovery, a left-sided, unilateral SCI (the C2Hx performed in the current study) profoundly affects the ipsilesional forelimb 135 – 138 . It is intuitive therefore that locomotion might depend more upon the contralateral, right limbs to compensate for left-sided deficits. In our study, clodronate treatment enhanced this compensatory increase, specifically by increasing the surface area used by the right hindpaw to contact the walkway during locomotion ( Figure 8Av ). Additional non-significant effects seemed to trend towards improving interlimb coordination ( Figure 8Bi , regularity index) and increasing hindpaw weight-bearing ( Figure 8.Biii ). However, carprofen treatment did not affect locomotor function after C2Hx ( Figure 9 ). In the interpretation of these results, we consider clodronate and carprofen-mediated analgesia. The CatWalk variable of Maximal Contact Maximal Intensity described in this study as a proxy measure of maximum weight-bearing ( Figure 8Bii – v ) is correlated with behavioral metrics of experimental hyperalgesia/allodynia such as Von Frey testing 139 . Additionally, animal models of unilateral cervical SCI have previously identified neuropathic mechanosensitive pain following injury 138 . While no statistical significance is present for the Maximal Contact Maximal Intensity variable in our study, it is intuitive to expect that altered sensation might play a role in not only this outcome measure, but multiple others measured by CatWalk. Though analgesia is a well-known and intended result of carprofen treatment following SCI, it is possible that the hematogenous monocyte-depleting effects of clodronate might also induce an analgesic effect in addition to neuroprotective mechanisms previously discussed in studies of contusion SCI 6 , 8 , 9 , 11 , 12 , 138 . In animal models, clodronate-mediated depletion of MDMs and spinal cord microglia can attenuate mechanical allodynia in experimentally induced neuropathic pain 140 , 141 . More specific to our field of interest, experimental clodronate treatment following thoracic contusion diminished spinal cord expression of a potentially pro-inflammatory pain mediator called bone morphogenetic protein 4 (BMP4) 12 . To experimentally evaluate the role which sensation plays in locomotion following the C2Hx and treatment with clodronate and carprofen, it will be necessary for future studies to conduct mechanosensory evaluation with Von Frey and thermosensitivity testing along with locomotion in future investigations. Additionally, histological comparison of locomotor muscular inflammation and atrophy between drug-exposed and control subjects would be insightful 142 . Preinjury, pre-treatment weight was significantly lower in subjects assigned to the saline-treated group in comparison to all three others ( Figure 3A ), despite randomized group assignments. However, locomotor and diaphragm electromyography outcomes do not scale by body mass and are unlikely to be substantially impacted. Since volume-related metrics of ventilatory function do significantly differ by subject body habitus, our intergroup pretreatment weight discrepancies emphasize the importance of utilizing weight-based normalization of tidal volume and minute ventilation parameters measured by WBP 28 , 143 – 146 . While daily body weight is a useful proxy for normalizing lung volume differences in healthy subjects, C2Hx injury and subsequent treatments likely influence ventilation and weight through distinct mechanisms, making post-injury weight a less reliable metric for ventilatory normalization—especially at early time points. Given these distinct influences, we normalized V T and V E to preinjury weight in this study to minimize the confounding effects of post-injury weight loss, which was also significantly impacted by treatment ( Figure 3B ). Weight loss is a common clinical and veterinary sign in conditions relating to bone resorption that require treatment with bisphosphonate drugs like clodronate 147 – 150 . As treatment improves the underlying cause of chronic illness, weight loss often is also attenuated. In contrast, our study’s clodronate-mediated weight loss suggests that bisphosphonate treatment did not address the pathology’s root cause in the same sense as in diseases of bone resorption. Thus, we suggest that clodronate’s effects on peripheral organs might have induced an increased general sense of malaise, reduced appetite, and led to the observed exacerbation of injury-induced weight loss. COX-1 inhibition can lead to gastrointestinal ulcer formation, reduced appetite, and weight loss 151 – 153 . Additionally, persons with traumatic SCI are at high risk for gastrointestinal ulcer formation and are prophylactically treated with proton-pump inhibitors as part of the clinical standard of care 154 – 159 . We propose that carprofen treatment may have exacerbated post-C2Hx weight loss in our rats through non-selective COX-1 activity, contributing to gastrointestinal pathologies that could mirror the ulcer formation commonly observed after clinical SCI. Future studies should investigate whether murine models of SCI show signs of gastrointestinal pathology akin to the clinical population. Finally, we must discuss potential sources of error within our study, including differential attrition rates, administration of supplemental fluids, and extravasation of tail vein injections between treatment groups ( Figure S1 – S6 ). Following statistical analysis, we found no evidence that our treatment groups underwent significantly increased attrition, received unequal fluids post-surgically, or were disproportionally affected by injection extravasation ( Figure S1 ). Across 131 regression analyses examining associations between the number of potentially extravasated injections and our respiratory and locomotor outcomes, only 8 reached nominal statistical significance ( Figures S2 – S6 ). We intentionally did not apply corrections for multiple comparisons, as our goal was to detect any possible influence of extravasation, even at the risk of identifying false positives. Moreover, statistically significant regressions were sometimes positive and sometimes negative, further suggesting the absence of a systematic effect. Collectively, these results indicate that clodronate injection extravasation is unlikely to have meaningfully influenced our study’s breathing and locomotor outcomes. 5. Conclusion In summary, systemic treatment with the translationally relevant drugs clodronate and the NSAID drug carprofen synergistically impaired, rather than improved breathing function following C2Hx. However, clodronate treatment paradoxically improved locomotor recovery, similar to that which has been previously established in published studies of more diffuse, caudal models of SCI. Future experimentation is needed to determine whether clodronate treatment negatively impacts breathing function across all SCI types and levels, if its effects are specific to high cervical laceration injury, and to discover the peripheral organ and/or centrally mediated mechanism underlying these divergent functional outcomes. Overall, this study advances understanding of the therapeutic potential of clodronate and carprofen and helps direct future preclinical research toward more effective translation of immunomodulatory therapies for improved treatment of SCI. Supplementary Material 1 NIHMS2119318-supplement-1.pdf (99.8KB, pdf) 2 NIHMS2119318-supplement-2.pdf (67.9KB, pdf) 3 NIHMS2119318-supplement-3.pdf (106.5KB, pdf) 4 NIHMS2119318-supplement-4.pdf (356.9KB, pdf) 5 NIHMS2119318-supplement-5.pdf (179.5KB, pdf) 6 NIHMS2119318-supplement-6.pdf (381.1KB, pdf) 7 NIHMS2119318-supplement-7.docx (21KB, docx) 8 NIHMS2119318-supplement-8.docx (45.3KB, docx) Highlights. Translational bisphosphonate and NSAID agents tested in experimental cervical SCI Contrary to hypothesis, bisphosphonate (clodronate) impaired ventilatory recovery NSAID (carprofen) treatment also worsened ventilatory outcomes Clodronate treatment improved compensatory locomotor function Divergent outcomes reveal treatment effects depend on organ system and injury level 6. Acknowledgements The authors thank Adam Bachstetter and the University of Kentucky Center for Applied Artificial Intelligence within the Institute for Biomedical Informatics for their help regarding use of the CLASSify tool in relation to CatWalk variables. Additionally, the authors thank Michael Sunshine for development of scripts to perform breath-by-breath analysis of whole-body plethysmography data, and initial guidance for CatWalk acquisition parameters. We also thank Mohit Patel, an Alilain lab undergraduate researcher, as well as Reena Kumari and Sajeev Kaur, Gensel lab members, whose willingness to lend their time and expertise to discuss experimental plans and techniques were highly valuable for our experimentation. Finally, the authors acknowledge the use of OpenAI’s ChatGPT (GPT-4, March 2025 version) for assistance in rephrasing select sentences and providing general guidance on some statistical analysis approaches. 9. Funding Information This work was financially supported by supported by the National Institutes of Health (R01NS116068 to John Gensel (JCG) and Warren Alilain (WJA); R21NS137256 to Meifan Chen (MC) and WJA; T32AA027488 to directors Mark Fillmore and Mark Prendergast, awarded to Aaron Silverstein) and a Pilot Grant from the Craig H. Neilsen Foundation (to MC and WJA). Footnotes Declaration of Interest Statement No competing interests of any kind, financial or otherwise, exist for this manuscript. Data from this manuscript was presented as an abstract and poster at the 2024 Kentucky Spinal Cord and Head Injury Research Trust and as an oral presentation at the June 2025 American Spinal Injury Association conference in Scottsdale, AZ. Furthermore, this manuscript was included with minor modifications as the third chapter of Aaron Silverstein’s PhD doctoral dissertation, planned to be submitted in May of 2025. Additionally, CatWalk data from the current manuscript’s saline treatment group was used as the C2Hx-injured group in a separate short communication manuscript describing locomotor deficits following C2Hx. The manuscript for that study is in preparation and will be cited appropriately once such a citation is available. 8. Authors’ Disclosure Statement No competing interests of any kind, financial or otherwise, exist for this manuscript. Data from this manuscript was presented as an abstract and poster at the 2024 Kentucky Spinal Cord and Head Injury Research Trust and as an oral presentation at the June 2025 American Spinal Injury Association conference in Scottsdale, AZ. Furthermore, this manuscript was included with minor modifications as the third chapter of Aaron Silverstein’s PhD doctoral dissertation, planned to be submitted in May of 2025. Additionally, CatWalk data from the current manuscript’s saline treatment group was used as the C2Hx-injured group in a separate short communication manuscript describing locomotor deficits following C2Hx. The manuscript for that study is in preparation and will be cited appropriately once such a citation is available 10. Transparency, Rigor, and Reproducibility This study was not pre-registered, but rigorous methodological standards were followed throughout. Group assignments were randomized and data collection and outcome assessments were performed by experimenters blinded to treatment groups wherever feasible. All statistical hypothesis testing was planned before data analysis. However, regressions examining relationships between possible extravasation and main study outcomes were added after hypothesis testing in the spirit of fully evaluating possible confounds. All statistical analysis was done in collaboration with a biostatistician. All animals and data points are fully accounted for, including missing data and attrition, and these are transparently reported in the manuscript. No replication or external validation studies are currently planned or ongoing to our knowledge. All raw data have been uploaded to the Alilain Lab space on the FAIR-compliant repository ODC-SCI.org, and will be made publicly available as soon as possible. Analysis scripts and metadata are available upon reasonable request to the corresponding authors. The study followed ARRIVE 2.0 guidelines for animal research reporting. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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