Buprenorphine Alters Microglia and Astrocytes Acutely Following Diffuse Traumatic Brain Injury.

Buprenorphine Alters Microglia and Astrocytes Acutely Following Diffuse Traumatic Brain Injury. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Buprenorphine Alters Microglia and Astrocytes Acutely Following Diffuse Traumatic Brain Injury. Jane Ryu, Phillip Stone, Sabrina Lee, Brighton Payne, Karen Gorse, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-138979/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Apr, 2021 Read the published version in Scientific Reports → Version 1 posted 8 You are reading this latest preprint version Abstract Background: Traumatic brain injury (TBI) is a common phenomenon, accounting for significant cost and adverse health effects. While there is information about focal pathologies following TBI, knowledge of more diffuse processes is lacking, particularly regarding how analgesics affect this pathology. As buprenorphine is the most commonly used analgesic in experimental TBI models, this study investigated the acute effects of the opioid analgesic buprenorphine (Bup-SR-Lab) on diffuse neuronal/glial pathology, neuroinflammation, cell damage, and systemic physiology. Methods : We utilized a model of central fluid percussion injury (CFPI) in adult male rats treated with a single subcutaneous bolus of Bup-SR-Lab or saline 15min post-injury. Microscopic assessments were performed at 1 day post-injury. Cell impermeable dextran was infused intraventricularly prior to sacrifice to assess neuronal membrane disruption. Axonal injury was assessed by investigating labeling of the anterogradely transported amyloid precursor protein. Neuroinflammation was assessed by analyzing Iba-1+ microglial and GFAP+ astrocyte histological/morphological features as well as cytokine levels in both regions of interest (ROIs). Myelin pathology was assessed by evaluating the expression of myelin basic protein (MBP) and the propensity of MBP+ myelin debris. Results : Acute physiologic data showed no difference between groups except for reduction in weight loss following cFPI in Bup treated animals compared to saline. There were no discernable differences in axonal injury or membrane disruption between treatment groups. Cytokine levels were consistent between Bup and saline treated animals, however, microglia and astrocytes revealed region specific histological changes at 1d following Bup treatment. Myelin integrity and overall MBP expression showed no differences between Bup and saline treated animals, but there were significant regional differences in MBP expression between the cortex and thalamus. Conclusions : These data suggest effects of Bup treatment on weight following CFPI and potential regional specificity of Bup-associated microglial and astrocyte alterations, but very little change in other acute pathology at 1-day post-injury. Overall, this preliminary study indicates that use of Bup-SR-Lab in preclinical work does have effects on acute glial pathology, however, longer term studies will be needed to assess potential effects of Bup treatment on more chronic pathological progressions. Cellular & Molecular Neuroscience Traumatic brain injury buprenorphine Bup-SR-Lab microglia astrocyte membrane disruption myelin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Traumatic brain injury (TBI) is an increasingly common phenomenon, with almost 2 million reported cases occurring annually in the US alone 1–3 . Overall, TBIs contribute to nearly 30% of all injury-related deaths, and account for significant healthcare cost and adverse health effects. Brain injury-induced pathology can be subclassified into focal and diffuse changes. Focal pathology of TBI results from the impact of the brain, and results in relatively homogenous sequelae of injury. Diffuse pathology, on the other hand, can be widely distributed throughout the brain in “pockets” of injury, and involves heterogenous cellular responses, ranging from diffuse axonal injury (DAI) and neuronal membrane disruption to neuroinflammatory changes and myelin pathology, making it difficult to track in the human population. Therefore, animal models of TBI are utilized for the rigorous assessments of TBI-induced pathology. These experimental TBI studies are being brought into greater alignment modeling the common data elements utilized in clinical TBI studies by groups such as the Federal Interagency Traumatic Brain Injury Research (FITBIR) data sharing group 4 making metanalysis of experimental studies much more feasible. As various models of experimental diffuse TBI require surgical intervention, guidelines suggest the use of analgesics 5,6 . The most commonly used analgesics in experimental studies are opioids. However, there are indications that opioids may alter neuronal survival and possibly inflammation 7–11 , and the effects of opioids on various other pathologies have not been rigorously tested. Because effects of analgesics on neuropathology and physiology are poorly described, it is unclear if their use confounds data. This uncertainty regarding possible secondary effects of opioid administration post-injury has led to a debate regarding the use of analgesics following TBI. Buprenorphine (Bup) is a semi-synthetic opioid derived from thebaine, one of six naturally occurring alkaloids of the opium poppy 10,12 . It is a partial agonist of the Mu receptor and an antagonist of the Kappa and Delta opioid receptors 12,13 . Due to these binding properties, Bup is commonly used in the treatment of opioid use disorder, the context in which the drug is most often studied 14,15 . Bup is also the most commonly used analgesic in pre-clinical animal models owing to the effectiveness of the sustained release formulation of Bup, Bup-SR-lab, in pain reduction over multiple days following a single subcutaneous administration 16–21 . While there are indications that Bup could affect cellular pathology 7,22,23 , the specific effects of Bup-SR-Lab on neuronal/glial pathology, neuroinflammation, cell damage, and physiology following brain injury are still unclear. Therefore, this study sought to determine the effects of Bup-SR-Lab on various acute diffuse pathologies precipitated by TBI. Specifically, physiological changes, neuronal membrane disruption, axonal injury, microglial and astrocyte alterations, cytokine expression and myelin changes were assessed at 1 day following diffuse central fluid percussion injury (CFPI) in adult male Sprague-Dawley rats treated subcutaneously with 1mg/kg Bup-SR-Lab or saline at 15min post-injury. Methods Animals Experiments were conducted in accordance with ARIVE guidelines and the Virginia Commonwealth University institutional ethical guidelines concerning the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University, which adhere to regulations including, but not limited to, those set forth in the “Guide for the Care and Use of Laboratory Animals: 8th Edition” (National Research Council). Overall, 12 adult (12 to 16-week-old; n=6/group) male Sprague-Dawley rats were used for this study. Animals were housed in individual cages on a 12-hour light-dark cycle with free access to food and water and full veterinary oversight. Surgical Preparation, Injury Induction, and Drug Administration Anesthesia was induced with 4% isoflurane in 30% O 2 /70% room air. Animals were then intubated and ventilated with 1.5%-2.5% isoflurane in 30% O­ 2 and 70% room air throughout the duration of the surgery, injury, and physiologic monitoring. Body temperature was maintained at 37 o C with a rectal thermometer connected to a feedback-controlled heating pad (Harvard Apparatus, Holliston, MA, USA). All animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). A midline incision was made followed by a 4.8 mm diameter circular craniectomy, which was positioned along the sagittal suture midway between bregma and lambda. The dura was left intact. A 2mm diameter burr hole was also drilled into the left parietal bone overlaying the left lateral ventricle (0.8 mm posterior, 1.3 mm lateral, and 2.5 to 3 mm ventral relative to bregma) through which a 25-gauge needle, connected to a pressure transducer and a micro infusion pump 11 Elite syringe pump (Harvard Apparatus) via PE50 tubing, was placed into the left ventricle. Appropriate placement of the infusion pump into the lateral ventricle was verified via a 2.3μl/min infusion of sterile saline within the closed fluid pressure system during needle placement 24,25 . The needle was held in the lateral ventricle for at least 5 minutes to record preinjury ICP; then the needle was slowly removed. Bone wax was used to seal the burr hole used for the ICP measurements before preparation for central fluid percussion injury (CFPI). The procedures used to induce CFPI were consistent with those described previously 24–27 . Briefly, a Luer-Loc syringe hub was affixed to the craniotomy site with dental acrylic (methyl methacrylate; Hygenic, Akron, OH, USA) that was applied around the hub, including the area overlying the sealed burr hole and allowed to harden. Animals were removed from the stereotaxic frame and placed on a raised platform for connection to the fluid percussion device, maintaining an unbroken fluid-filled system from the intact dura through the cylinder, via a Leur-Loc adaptor. During injury the investigator supported the animal’s body on the platform but did not hold the head allowing the Leur-Loc mechanism to maintain connection between the injury hub and fluid percussion device. To induce a mild-moderate CFPI a pendulum was released onto the fluid-filled cylinder of the FPI device, producing a pressure pulse of 2.05±0.10 atmospheres for ~22.5msec (table 1), which was transduced through the intact dura to the CSF. The pressure pulse was measured by a transducer affixed to the injury device and displayed on an oscilloscope (Tektronix, Beaverton, OR, USA). Immediately after the injury, animals were reconnected to the ventilator and physiologic monitoring devices. The hub, dental acrylic, and bone wax were removed en bloc and Gelfoam was placed over the craniectomy/injury site. The animal was then replaced in the stereotaxic frame, and the ICP probe was reinserted into the lateral ventricle, as described above, for postinjury ICP monitoring. Immediate post-injury physiology was recorded for 15min after CFPI followed by subcutaneous administration of either 1mg/kg Bup SR-Lab or saline. The surgeon randomly selected a pre-filled blinded syringe that was administered by another investigator to avoid inadvertent unblinding of the surgeon due to the difference in viscosity of the solutions that might introduce bias that could influence animal care. One hour following injury the scalp was sutured and treated with lidocaine and triple-antibiotic ointment. Rats were then allowed to recover and were returned to clean home cages. Physiologic Assessment Heart rate, respiratory rate, and hemoglobin oxygen saturation were monitored via a hindpaw pulse oximetry sensor (STARR Life Sciences, Oakmont, PA, USA) for the duration of anesthesia, except during the induction of injury. Intracranial pressure (ICP) was measured intraventricularly, as described above. All physiologic measurements were recorded using a PowerLab System (AD Instruments, Colorado Springs, CO, USA). All animals maintained systemic physiological homeostasis throughout the experiment (i.e., heart rate>200 beats per min and oxygenation>90%; Table 1; Figure 1). Changes in ICP following CFPI were noted in both groups, particularly at 1d post-injury (Table 1; Figure 1C). Recovery time following CFPI (the time from withdrawal of inhaled anesthetic to first movement) and weight loss (percent reduction in animal weight from pre-injury to 1d post-injury) were also assessed (Table 1; Figure 1). Tracer Infusion At 1d post-injury rats were anesthetized with 4% isoflurane in 30% O 2 /70% room air followed by maintenance dose of 2% isoflurane in 30% O 2 /70% room air via a nose cone. Animals were secured into a stereotaxic device and the incision from the previous day was reopened by removing the sutures. The ICP needle was filled with 0.7mg/17μl 10kDa biotinylated dextran and placed into the left lateral ventricle with continuous ICP monitoring, as described above. The ICP at 1d post-CFPI was measured for 15min following needle placement then the dextran was infused at a rate of 1.3μl/min with continuous ICP monitoring. The tracer was allowed to diffuse for 2h before animals underwent transcardial perfusion, as described below. Tissue Processing At 1d post-CFPI 2hr following tracer infusion, anesthetized rats were overdosed with Euthasol euthanasia-III solution (Henry Schein, Dublin, OH, USA) followed by transcardial perfusion with cold 0.9% saline. As was described previously 25 , both fresh and fixed brain tissue was collected from each animal. A tissue core of the right lateral neocortex and thalamus/midbrain was taken for molecular assessments prior to transcardial fixation with 4% paraformaldehyde/0.2% glutaraldehyde in Millonig’s buffer (136 mmol/L sodium phosphate monobasic/109 mmol/L sodium hydroxide) for immunohistochemical analysis of the left side of the brain. After transcardial perfusion, the left side of the brain was removed and postfixed for >72h. Postfixed brains were sectioned coronally in 0.1 mmol/L phosphate buffer with a vibratome (Leica, Banockburn, IL, USA) at a thickness of 40 μ m from bregma to ∼4.0 mm posterior to bregma. Sections were collected serially in 12-well plates and stored in MIllonig’s buffer at 4 o C. All quantitative analyses were performed at least 1 mm posterior to the needle track used for ICP monitoring. The well from which sections would be taken for analysis was selected via a random number generator (1-12) and the first 4 sections with visible hippocampus were taken representing serial sections throughout the rostral-caudal extent (1.8 mm±0.2 mm to 3.8 mm±0.2 mm posterior to bregma, each 480μm apart). Histologic analyses were performed on the left lateral somatosensory cortex restricted to layers V and VI extending from the area lateral to CA1 to the area lateral to CA3 of the hippocampus and entire left hemi-thalamus extending from the midline and dorsal surface of the thalamus to the reticular nucleus and zona inserta of the thalamus laterally and ventrally. Assessment of Cell Damage To evaluate the numbers of damaged cells in the cortex and thalamus of rats following injury and vehicle or Bup treatment, four sequential, randomly selected sections per animal were stained with hematoxylin and eosin (H&E) and assessed as described previously 24,25,28 . Briefly, tissue was mounted on gelatin-coated slides before dehydration and rehydration. Rehydrated tissue was incubated in Gills hematoxylin (Leica Biosystems, Buffalo Grove, IL, USA) followed by bluing agent (Leica Biosystems) and three dips in 0.25% eosin Y/0.005% acetic acid/95% ethanol before sections were cleared through increasing concentrations of ethanol and cover-slipped with Permount (Thermo Fisher Scientific, Waltham, MA, USA). Sections were visualized using a Nikon Eclipse 800 microscope. Assessments were done on the left side of the cortex and thalamus for each section. The number of damaged neurons, delineated by eosinophilic cytoplasm and condensed nuclei, in the entire left lateral neocortex and thalamus was counted by two independent investigators blinded to the animal group and averaged for each animal and each group. Data is reported as the number of damaged cells/region of interest (ROI). Assessment of Axonal Injury To quantify axonal injury, immunohistochemistry targeting amyloid precursor protein (APP) was performed. Sections were immunolabeled, as previously described 24,29 . Tissue was blocked and permeabilized in 5% normal goat serum and 1.5% triton followed by overnight incubation with a primary rabbit antibody against the C terminus of β -APP (Cat. #51-2700, 1:700, Life Technologies). Secondary antibody, biotinylated goat anti-rabbit IgG (Cat. #BA-1000, 1:1000, Vector Laboratories, Burlingame, CA, USA) was then incubated for 2h at room temperature. The sections were subsequently incubated in avidin biotinylated enzyme complex using the Vectastain ABC kit (Vector Laboratories) followed by visualization with 0.05% diaminobenzidine/0.01% H 2 0 2­ /0.3% imidazole/phosphate-buffered saline. The tissue was mounted, dehydrated, and cover-slipped. Visualization of APP-labeled axonal swellings was performed using a Nikon Eclipse 800 microscope (Nikon, Tokyo, Japan) equipped with an Olympus DP71 camera (Olympus, Center Valley, PA, USA). The total number of APP+ axonal swellings in the entire region of interest (the left lateral neocortex layers V and VI or the left thalamus) for each section was counted by eye by an investigator blinded to animal group. Immunofluorescence To identify microglia, astrocytes, and myelin, fluorescent immunohistochemistry against the calcium binding protein, Iba-1 (microglia), glial fibrillary acidic protein, GFAP (astrocytes), or myelin basic protein, MBP (myelin) was done. Briefly, 40mm thick coronal sections were blocked and permeabilized in 1.5% triton and 5% normal goat serum followed by overnight incubation with primary antibody rabbit anti-Iba-1 (Cat. #019-19741, 1:1000, Wako; Osaka, Japan), mouse anti-GFAP (Cat.#MAB3402, 1:1000; Millipore Burlington, MA, USA) or mouse anti-myelin basic protein (Cat #808401; 1:1000; BioLegend, San Diego, CA, USA) overnight followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Cat.# A11034, 1:700; ThermoFisher Scientific) or Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (Cat.# A-11031, 1:700; ThermoFisher Scientific). Tissue was mounted using Vectashield hardset mounting medium with Dapi (Cat.#H-1500; Vector Laboratories). Immunolabeling for all tissue was done at the same time to reduce run-to-run variability. All image acquisition settings were held consistent between groups for each region of interest (left lateral neocortex layers V and VI or the left thalamus) and imaging was done by an investigator blinded to animal group. Dapi nuclear labeling was used to verify focus and restriction within the regions of interest prior to image acquisition. Assessment of Neuronal Membrane Disruption Consistent with previous studies, we assessed the potential for neuronal membrane disruption via the utilization of tagged 10 kDa dextran 24,25,30 , which are impermeable to cells with intact membranes. Cells containing dextran, therefore, indicate membrane disruption. Immunolabeling for all tissue was done at the same time to reduce run-to-run variability. Sections were blocked with 5% normal goat serum, permeabilized with 1.5% triton, and immunolabeled with primary antibodies mouse anti-NeuN (Cat. #MAB377, 1:500, Millipore), to identify neurons, and goat anti-biotin (Cat #31852, 1:2,000, ThermoFisher Scientific; Waltham, MA, USA) to identify dextran. Secondary antibodies Alexa-fluor 568-conjugated donkey anti-goat IgG (Cat. #A11057, 1:700, ThermoFisher Scientific) and Alexa-fluor 488-conjugated goat anti-mouse IgG (Cat. #A11001, 1:700, ThermoFisher Scientific) were then incubated and the tissue was mounted with Vectashield hardset mounting medium with DAPI (Cat.#H-1500; Vector Laboratories). Sections were imaged by confocal microscopy using a Zeiss LSM 700 System (Carol Zeiss, Oberkochen, Germany). Confocal images of the left neocortex layers V and VI were taken at x 40 magnification in a systematically random fashion by an investigator blinded to animal group using DAPI labeling to verify focus and NeuN label to verify location within the region of interest. Image acquisition settings were held constant for comparable regions for all groups analyzed. Analysis of NeuN + neurons containing the cell-impermeable dextran was performed by an investigator blinded to animal group using the ImageJ colocalization finder plugin and traditional cell counting. Dextran containing neurons were quantified for each image and averaged for each animal. Assessment of Microglial Activation Four sections/animal labeled with Iba-1 were imaged using an Olympus DP71 camera (Olympus, Center Valley, PA, USA) and were analyzed using FIJI/ImageJ as follows. All cells in which both the cell body and process network were in focus were marked in each image and a subset (n=5/image) were randomly selected using a random number generator for further morphological analysis. Each randomly selected cell was individually analyzed for process number, number of branch end/terminal points, and maximum process segment length using the skeleton analysis tool in ImageJ. A complexity index was also calculated for each microglia using the formula, complexity index= number of processes/number of end points, with a lower number indicating reduced process complexity. The soma of each cells was also circumscribed to assess the perimeter of the cell body through ImageJ particle analysis. All data was recorded by an investigator blinded to animal group and averaged for each image. Individual microglia were considered ns. Assessment of Astrocyte Activation Three randomly selected images in a randomly selected section were taken at 20x magnification under consistent microscope settings for each region of interest (left lateral neocortex layers V and VI or the left thalamus) using a Keyence BZ-X800 microscope with section scanning on to reduce background (Keyence Corporation of America, Itasca, IL, USA). Images were processed with background subtraction and automatic thresholding to generate masks of GFAP+ astrocytes. All cells within the mask were added to the Region of Interest Manager in FIJI/ImageJ. Measurements of the number of astrocytes/image, cellular area and circularity of individual astrocytes and the percent of GFAP+ astrocyte coverage/image were assessed. All data was analyzed by an investigator blinded to animal group and averaged for each image and each animal. Individual animals were considered ns. Assessment of Myelin Integrity Four sections per animal with 6 images/section for the cortex or 4 images/section for the hemi-thalamus were imaged at 40X magnification in a systematically random fashion for each ROI using a Keyence BZ-X800 microscope with section scanning on to reduce background (Keyence Corporation of America). All images were captures and analyzed by an investigator blinded to animal group using the DAPI label to verify focus and location within the ROI. Image acquisition settings were held constant for comparable regions (cortex or thalamus) for all groups analyzed. Analysis of intact myelin fibers and myelin debris was performed using the Analyze particle plugin in FIJI/ImageJ (National Institutes of Health) with size and circularity parameters for object differentiation. The parameters used to determine myelin debris were circularity=0.3-1.0 and particle size=0.5-10 µm 2 . To assess myelin fibers the analysis settings were as follows, circularity= 0.0-0.1 and particle size= 25-infinity µm 2 . The average total area covered by intact myelin fibers or myelin debris was quantified for each image and averaged for each animal. Individual animals were considered ns. Quantification of Protein Expression Tissue from the right lateral neocortex and thalamus was homogenized in NP40 Buffer (150mM NaCl, 50 mM Tris pH 8.0, 1% Triton-X) and protease inhibitor cocktail (AEBSF 10.4mM, Aprotinin 8μM, Bestatin 400μM, E-64 140μM, Leupeptin 8μM, Pepstatin A 150μM, Cat#: P8340, Sigma, Saint Louis, MO, USA). Protein concentration was determined using a bicinchoninic acid assay in accordance with manufacturer’s instructions (Cat#23225; ThermoFisher) and quantified on a PHERAstar Spectrophotometer (BMG Labtech, Cary, NC, USA). For assessment of cytokine expression, cortical or thalamic protein homogenates were sent to Quansys Bioscience (Logan, UT, USA) for cytokine analysis of rat IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-10, IL-12, IFNy, and TNFa. Three replicates were run for each sample and the means of the replicates were used for each animal. Cytokine concentration (in pg) was normalized to total protein concentrations (in mg) for each sample. Samples in which there was no detectable amount of cytokine were set to 0pg/mg for analysis (IL-1b Thalamus saline n=3, Thalamus Bup n=1; IL-12 Thalamus saline n=4, Thalamus Bup n=4) after verifying acceptable (>0.5mg/ml) total protein concentrations. To analyze myelin basic protein (MBP) expression, Western blotting was performed. Protein (15 ug) was boiled for 10 min in 2x Laemelli loading buffer and run at 200 volts for 30 min on Mini-PROTEAN TGX Stain-free 4-20% precast polyacrylamide gels (Cat #4568096; BioRad, Hercules, CA, USA). Protein was transferred onto 0.2 um PVDF membranes using a Transblot Turbo transfer system (Bio-Rad) under the low molecular weight manufacturer settings (2.5 Amps, 25 Volts for 5 min). Western blotting was done on an iBind flex apparatus (Invitrogen) using primary antibodies rat anti-myelin basic protein (1:1000, Cat #MAB386; Millipore Sigma) and mouse anti-actin (1:4000, Cat #66009-1-Ig; Proteintech; Rosemont, IL, USA) followed by anti-rat-HRP secondary antibody (1:5000; Cat#112-035-003; Jackson Laboratories, West Grove, PA) and anti-mouse-HRP secondary antibody (1:5000, Cat #115-035-003; Jackson Laboratories; West Grove, PA). Chemiluminescent images were taken on a ChemiDoc imaging system (BioRad). Densitometric analysis was done in ImageJ (National Institutes of Health) for actin and total MBP expression, as well as individual MBP isoform expression. Total MBP expression was measured by taking the densities of each individual MBP isoform band and adding them to get the total sum. This method reduced biasing of the analysis by the variable degrees of white space between the MBP isoform bands in each run. MBP was then normalized to actin and to sham controls. All western blots were run in triplicates on three separate gels to reduce run-to-run variability potentially biasing the results. Statistical analysis A Shapiro-Wilk test for normality of the data was done prior to utilizing non-parametric statistics for data that was not normally distributed. The number of animals to be assessed for each group was determined by power analysis using previous data, an alpha=0.05 and a power of 80%. One-way or two-way ANOVA were done with Bonferroni post-hoc corrections for multiple comparisons. Statistical significance was set to a p value <0.05. Data are presented as mean± standard error of the mean (SEM) unless otherwise indicated. Results Post-injury weight loss was decreased with Bup-SR-Lab treatment As has been well characterized previously, CFPI (2.05±0.1 atmospheres) did not generate gross tissue pathology 24,25,27,28 . While there was apparent sub-arachnoid bleeding at 1d post-injury, there was no apparent damage to the underlying cortex. Neither saline nor Bup treated animals demonstrated indications of contusion, hematoma formation, or overt cortical compression. Body temperature was maintained at 37°C with a feedback loop thermoregulatory system connected to a rectal thermometer to avoid potential confounds of hypothermia in either group. As drug treatment was both randomized and blinded there was little possibility to fully match injury metrics between treatment groups, however, there was no difference in pre-injury weight (One-way ANOVA F 1,12 =0.73, p=0.411), injury intensity (One-way ANOVA F 1,12 =0.68, p=0.427), or injury duration (One-way ANOVA F 1,12 =0.013, p=0.913) between groups (Table 1). While recovery time (time from withdrawal from anesthesia to first sporadic movements) was higher in the Bup treated group on average, there was substantial variability in this group and ultimately this difference was not significant (Table 1; One-way ANOVA F 1,12 =2.82, p=0.124). Table 1: Physiological readouts in buprenorphine and saline treated animals prior to and following cFPI. g= grams, atm=atmospheric pressure, BPM=beats per minute, D ICP 10min-1d=change in intracranial pressure from 10min post-injury to 1d post-injury. Data presented as mean (standard deviation). Saline Bup Pre-injury Weight (g) 509 (87) 545 (61) Injury Intensity (atm) 2.05 (0.08) 2.01 (0.10) Injury duration (msec) 22.34 (0.64) 22.37(0.21) Recovery Time (min) 19.6 (7.4) 79.9 (78.9) D ICP 10min-1d 8.51 (6.74) 7.51 (4.04) To evaluate the potential effects of Bup on physiology, blood oxygen saturation, heart rate, and intracranial pressure were assessed prior to injury and at 1h and 1d following CFPI. Animal weight was also assessed prior to and 1d following CFPI to explore potential effects of Bup on post-injury weight loss. There was no discernable difference in blood oxygenation or heart rate between saline and Bup treated animals at any time point assessed (Figure 1 A&B). Intracranial pressure was also comparable between treatment groups at all time points measured (Figure 1C; Two-Way ANOVA F 1,22 =2.37; p=0.138). A significant increase in intracranial pressure of about 8mmHg from 1h to 1d post-injury was, however, noted in both treatment groups (Two-Way ANOVA F 1,22 =5.50; p=0.028). This change in intracranial pressure was also consistent between saline and Bup treated groups (Table 1). Weight loss, however, was significantly reduced in the Bup treated group (3.36±0.72% loss from pre-injury weight) as compared to the saline treated group (6.53±0.78% loss from pre-injury weight; One-way ANOVA F 1,11 =8.81; p=0.013). This difference in weight loss was not correlated to pre-injury weight (spearman Rho=0.14, p=0.648). Treatment with Bup-SR-Lab did not alter neuronal somatic or axonal injuries As CFPI in rats produces significant and various cellular pathology in the lateral neocortex and thalamic domains 24,27 , these regions of interest were the focus of all subsequent pathological assessments. Overt acute cellular damage/death was assessed using H&E staining. Any cell that demonstrated a heterochromatic nucleus and eosinophilic cytoplasm was considered to be damaged and/or undergoing early stages of cell death 25,27,28 (Figure 2a). There were very few damaged/dead cells in either the cortex (Figure 2 A-C; One-way ANOVA, F 1,10 =0.51, p=0.493) or the thalamus (Figure 2 D-F; One-way ANOVA F 1,10 =3.18, p=0.105) of either saline or Bup treated animals and no significant difference between groups was detected for either ROI. Diffuse axonal injury (DAI) is a hallmark of mild and/or diffuse TBI and is the leading pathological indicator of injury magnitude following a brain injury 31–33 . Therefore, the total number of amyloid precursor protein positive (APP+) axonal swellings, indicative of DAI-mediated axonal transport dysfunction, was assessed in both the lateral cortex and the thalamus 1d following CFPI and either saline or Bup treatment. While notable DAI was visible in both saline treated (cortex=17.10±3.39, Thalamus=89.33±35.50 APP+ swelling/ROI) and Bup treated (cortex=17.15±3.03, Thalamus=82.71±16.79 APP+ swelling/ROI) animals, the degree of DAI was indistinguishable between the two groups in either the cortex (Figure 3A-C; F 1,10 =1.4x10 -4 , p=0.990) or the thalamus (Figure 3D-F; One-way ANOVA F 1,10 =0.028, p=0.869). Finally, as somatic neuronal damage, in the form of neuronal membrane disruption, is present acutely following both diffuse and focal TBI, and as this pathology is exacerbated by secondary insults, we investigated the potential effects of Bup on neuronal membrane disruption 24,25,28,34–36 . As we and others have demonstrated previously, tagged 10kDa dextran, which is normally excluded from intact membranes, are a reliable way to evaluate neuronal plasmalemmal disruptions 24,25,28,35,37,38 . However, this method only allows assessment of the cortex, due to the variability of dextran diffusion within the thalamic domain. As found with axonal injury, there was no difference between saline treated and Bup treated groups in regard to the percent of neurons demonstrating membrane disruption 1d following CFPI (Figure 4; One-way ANOVA F 1,10 =0.192, p=0.671). Buprenorphine treatment alters microglia and astrocytes in a region-specific manor acutely following diffuse TBI. To assess the potential effects of Bup on acute inflammatory responses in the brain cytokine protein levels were assessed for each ROI in saline and Bup treated animals at 1d post-CFPI. Levels of IL-1a, IL-2, IL-6, IFNy, and TNFa were consistently below detectable limits for nearly all samples tested regardless of treatment group. Concentration of IL-1b, IL-4, IL-10, and IL-12, however, were measurable for the majority of samples tested. There did appear to be a regional difference in expression of IL-1b (Two-way ANOVA; Region F 1,16 =22.53, p=2.19x10 -4 ), IL-4 (Two-way ANOVA; Region F 1,16 =38.53, p=1.3x10 -5 ), and IL-10 (Two-way ANOVA; Region F 1,16 =35.75 p=1.9x10 -5 ) with consistently more cytokine expression in the cortex as compared to the thalamus (Table 2). However, there was no discernable treatment effect in either region for any of the cytokines evaluated (Two-way ANOVA; Treatment IL-1b F 1,16 =0.08 p=0.787; IL-4 F 1,16 =0.63 p=0.439; IL-10 F 1,16 =0.312 p=0.584; IL-12 F 1,16 =0.619 p=0.443). Table 2: Cytokine concentrations in cortex and thalamus of saline and buprenorphine treated animals at 1d following CFPI. Data presented as mean (SD) pg of cytokine per mg of total protein. Regional differences: # p<0.05 compared to cortex. Saline Buprenorphine Cortex Thalamus Cortex Thalamus IL-1b pg/mg 14.40 (4.44) 0.36 (0.50) # 14.40 (11.45) 2.29 (2.29) # IL-4 pg/mg 12.78 (6.23) 1.13 (1.15) # 12.97 (2.51) 3.63 (3.29) # IL-10 pg/mg 17.12 (7.71) 4.36 (2.77) # 18.69 (3.79) 5.23 (3.83) # IL-12 pg/mg 36.93 (23.04) 19.64 (43.93) 33.41 (28.87) 2.84 (6.35) To further investigate the potential that more subtle changes in neuroinflammation were occurring acutely following Bup treatment, microglia and astrocyte morphologies were analyzed. Alterations in microglial morphology indicative of an altered activation state (larger soma size, fewer process endpoints, shorter processes, and reductions in overall cell complexity 29,39,40 ) were assessed at 1d following CFPI in the cortex and thalamus of animals treated with saline or Bup. Microglia within the thalamic domain demonstrated approximately 11 process endpoints/cell (saline=11.38±0.25; Bup=11.39±0.25), with somal perimeters of about 30µm 2 (saline=29.20±0.49µm 2 ; Bup=30.01±0.64µm 2 ) and a maximum process segment length of around 20µm (saline=19.83±0.42µm; Bup=20.95±0.50µm) as well as an average cell complexity index of ~1.6 (saline1.606±0.01=; Bup=1.614±0.01 arbitrary units). There were no significant differences in any of these metrics between thalamic microglia in saline vs. Bup treated groups (Figure 5). Cortical microglia, however, demonstrated significant alterations in Bup treated animals as compared to saline. Cortical microglia in Bup treated animals had increased somal sizes (One-way-ANOVA, F 1,2627 =19.2, p=2.6x10 -12 ; cortex saline vs. Bup p=2.3x10 -5 ) and reduced process network complexity/microglia (One-way-ANOVA F 1,2627 =17.85, p=1.82x10 -11 ; cortex saline vs. Bup p=0.019) compared to saline treated cortical microglia (Figure 5). All microglial morphological metrics demonstrated significant region-specific differences in which the cortex displayed reduced numbers of process endpoints (Two-way-ANOVA; Region F 1,2627 =64.89, p=1.19x10 -15 ), larger soma sizes (Two-way-ANOVA; Region F 1,2627 =51.10, p=1.13x10 -12 ), shorter maximum process length/cell (Two-way-ANOVA; Region F 1,2627 =19.67, p=1.0x10 -5 ), and decreased process network complexity per microglia compared to the thalamus in either saline or Bup-treated animals (Two-way-ANOVA; Region F 1,2627 =44.05, p=3.87x10 -11 ; Figure 5). There was a significant interaction between drug treatment and region for the microglial complexity index (Two-way-ANOVA Region*Treatment F 1,2627 =6.15, p=0.013). As opposed to microglia, cortical GFAP+ astrocytes, did not appear significantly altered 1d following TBI and Bup treatment as compared to saline treated animals (Figure 6). There were slight trends toward higher numbers of astrocytes/image (Two-way-ANOVA; Treatment F 1,16 =3.31, p=0.09) covering a greater percentage of the image (Two-way-ANOVA; Treatment F 1,16 =4.11, p=0.06) between saline and Bup treatment, however, neither of these trends were statistically significant. While astrocyte cell size was consistent between saline and Bup treatment groups in the cortex, thalamic astrocytes demonstrated significantly greater cell size in the Bup-treated animals compared to the saline treated animals at 1d post-injury (Two-way-ANOVA; Treatment F 1,16 =8.12, p=0.01; Figure 6G). There were also significant regional differences in the morphology of astrocytes in the control animals, with more astrocytes (Two-way-ANOVA; Region F= 1,16 =9.49, p=0.007) covering a larger proportion of the image (Two-way-ANOVA; Region F= 1,16 =8.43, p=0.01) in the thalamus of saline-treated control animals compared to the cortical astrocytes. These regional differences were not maintained following Bup treatment (Figure 6). The size of astrocytes, however, was (Two-way-ANOVA; Region F= 1,16 =24.04, p=1.59x10 -4 ; Figure 6G). The circularity of astrocytes was also consistent between treatment groups and ROIs (Figure 6H). There were no interactions between region and treatment for any metrics. Treatment with Buprenorphine altered myelin basic protein (MBP) isoforms but not overall MBP expression In order to assess the effect of Bup on myelin pathology at 1d post-CFPI, immunohistochemical labeling and Western blotting were performed to evaluate myelin integrity and the expression of myelin basic protein (MBP). There was no significant difference in the total number of intact myelin fibers (One-way-ANOVA; Region F= 1,16 =1.224, p=0.344) or myelin debris (One-way-ANOVA; Region F= 1,16 =0.558, p=0.653) between ROI or drug treatment groups (Figure 7E). Furthermore, overall MBP expression did not appear to be significantly altered with Bup treatment in either region (One-way-ANOVA; Region F= 1,20 =0.064, p=0.803). There did appear to be statistically significant regional differences, however, where overall expression of MBP was higher in the cortex than the thalamus, regardless of drug treatment group (One-way-ANOVA; Region F= 1,20 =33.576, p=0.000). As the different MBP isoforms are linked to different developmental stages of myelination 41 , the individual MBP isoforms (21.5 kDa, 18.5 kDa, 17.2 kDa, and 14.0 kDa) were also analyzed. Similar to analysis of the overall expression of MBP, the expression of individual MBP isoforms was not statistically different between Bup and saline treatment groups (Two-way ANOVA; Treatment 21.5 kDa: F= 1,20 =0.054, p=0.819, 18.5 kDa: F= 1,20 =0.127, p=0.727, 17.2 kDa: F= 1,20 =0.001, p=0.982, and 14.0 kDa: F= 1,20 =1.85, p=0.673; Figure 7G). There were, however, statistically significant regional differences for all isoforms (One-way ANOVA; 21.5 kDa: F= 1,20 =12.410, p=0.000, 18.5 kDa: F= 1,20 =6.363, p=0.005, 17.2 kDa: F= 1,20 =17.202, p=0.000, and 14.0 kDa: F= 1,20 =5.364, p=0.010) in which the expressions of all isoforms were higher in the cortex of either saline or Bup treatment group compared to the thalamus (Figure 7G). Discussion Buprenorphine (Bup) is a semi-synthetic opioid which is a long-lasting and very effective partial agonist of the Mu opioid receptor (MOR) and an antagonist of the Kappa opioid receptor (KOR) and at higher doses the delta opioid receptor (DOR) 10,12 . The efficacy of low dose Bup in blocking the agonist effects of other opioids while also reducing withdrawal symptoms has made it a primary treatment for opioid use disorder 14 . Additionally, as there is a highly effective slow-release formula of Bup, that effectively alleviates pain for multiple days following a single subcutaneous injection, Bup is commonly used in experimental studies as a post-operative analgesic 16,19–21 . To our knowledge there has only been one study evaluating the potential effects of Bup following CNS injury 42 . While this study found no differences in focal pathology following spinal cord injury, there have been no studies systematically assessing the potential effects of Bup-SR-Lab on the acute diffuse pathology produced by CNS injury. As the potential for analgesic-induced alterations on pathological progression give some investigators pause, the current study evaluated the effects of Bup-SR-Lab on acute physiological and diffuse pathological changes following TBI. Although in previous studies Bup demonstrated physiological effects, such as respiratory depression and bradycardia 43 , we found that subcutaneous treatment with 1mg/kg Bup 15 min post-injury appeared to have no effect on systemic physiology. Specifically, heart rate and hemoglobin oxygen saturation were comparable between saline and Bup treated animals (Figure 1). While all animals in the current study were ventilated to maintain consistent respiratory rates, previous, studies found that Bup could act as a respiratory depressant. One study found that when rats were given a dose of Bup ranging from 1mg/kg to 3mg/kg respiratory rates decreased in a dose-dependent fashion 44 . Another study found that rats treated with 1.4mg/kg, 4.3mg/kg or 8.6mg/kg doses of Bup displayed a ceiling in Bup-induced respiratory depression after nearly 2 h of Bup infusion 45 . In another study, arterial pH and PaCO 2 were not altered in rats treated with 1.2mg/kg Bup-SR but did demonstrate lower arterial oxygen saturation compared to saline controls, indicating potential respiratory alterations 19 . This ceiling phenomenon, where respiratory depression reached its apparent maximum effect regardless of drug dose, is potentially attributed to Bup’s partial agonism at the MOR and may have prevented rapid changes in respiratory rate, as well as changes involving neural systems and behavioral processes 46 . While all animals lost weight following TBI, animals treated with Bup demonstrated better maintenance of their body weight within the first day following CFPI compared to the saline control group (Figure 1). In a previous study using Bup-SR, weight was measured within the first 4 days after 0.3-4.5mg/kg doses were given to rats. Rats that were given 1.2mg/kg doses of Bup-SR maintained their weight but rats given 0.3mg/kg and 4.5mg/kg of Bup-SR gained or lost weight, respectively 16 . This indicates that there may be a dose-regulated response to Bup in regard to weight change in rats. Another study examining rabbits found that treatment of 0.03mg/kg of Bup every 12 h for 48 h 1d post-operatively experienced immediate weight loss post-operatively that sustained over the next four days, however, the rats displayed a slow return to baseline body weight by day 5 post-operatively 47 . The reduced weight loss we observed at 1 day-post-injury may be attributed to the effects of Bup on gastrointestinal motility rather than suppressed appetite 48 . In a study examining the effects of 0.05mg/kg buprenorphine every 12 h for 48 h on rabbits found that buprenorphine induced gastrointestinal hypomotility of gut and delayed the passage of feces. Wheel running activity alters not only energy but food intake, neural systems involving stress response, and behavioral processes. A study found that voluntary wheel running activity was significantly lower at 24 h with Bup treatment but returned to baseline by 48 h 19 , which could impact body weight. The weight retention could also be mediated by the KOR. Kappa-opioid activation is known to cause stress, which is highly linked to weight loss 49 , but Bup acts as an antagonist of the KOR, which could reduce the effect of KOR-mediated weight loss. However, we were unable to find any studies investigating the association between KOR-activation and weight changes. Finally, while our study did not inspect the metabolism effects of Bup, it is known that Bup partially metabolizes to norbuprenorphine in the liver and both compounds are excreted as glucuronides. They then undergo enterohepatic circulation, where the drug and its metabolites can remain in intestinal circulation for days 50 . One study investigating Bup glucuronide metabolites discovered that two glucuronide metabolites of Bup are pharmacologically active 51 . This suggests that even as Bup is broken down in order to be eliminated from the body, its metabolites are still functioning and could have effects on weight loss at longer time points than the current study investigated. Although both treatment groups presented DAI, there were no discernable differences in axonal injury between groups, indicated by the total number of APP+ axonal swellings in either the thalamus or cortex. There were also no significant indications of cell damge/death 1d post-CFPI in either region regardless of Bup treatment. , a previous study found that Bup administration prevented neuronal death and protected neurons in the medial and lateral regions of the thalamic reticular nucleus following resuscitation from cardiac arrest 52 . This region-specific finding was interesting as MORs are present in the medial and lateral regions of the thalamic reticular nucleus but not in the central regions 52 . This suggests that Bup may selectively protect regions in a MOR-mediated fashion. As the CFPI model used in our study does not precipitate cell death post-injury in either the cortex or the thalamus 27,53–55 , it is unsurprising that Bup’s potential neuro-protective effects were not seen in the current study. Acute neuroinflammation, however, does occur following CFPI as it does in human TBI 29,54,56–60 . The primary cellular representatives for neuroinflammatory changes are microglia and astrocyte activations. The neuroinflammatory response of activated microglia and astrocytes occurs on a spectrum ranging from a pro-inflammatory phenotype, which is characterized by neurotoxic properties and release of neuroinflammatory cytokines, to an anti-inflammatory phenotype, which is characterized by the release of neurotrophic factors and anti-inflammatory cytokines that promote repair 61 . This correlates with the findings of higher cytokine expression (IL-1b, IL-4, IL-10, and IL-12) in the cortex, but our data found no discernable differences between treatment groups within either brain region analyzed. Corroboratively, studies have shown that animals treated with Bup, do not display systemic immunosuppression, which is common for MOR agonists, such as fentanyl 62 . One study, however, found higher levels of cytokines, such as IL-10 and TNFa, in the serum of animals treated with Bup compared to the control animals 63 , suggesting a potentially unique role of Bup on neuroinflammation as compared to other opioids. Another study recently found increased expression of IL-1b in the hippocampus of aged rats treated with morphine 64 weeks following surgical procedures, indicating that there might also be neuroinflammatory changes at later time points, which this study would not have captured. These possibilities will need to be further assessed at later time points following diffuse TBI, when neuroinflammatory responses are more robust 39,56 . Microglial and astrocyte activation can also be assessed via investigation of the morphological remodeling each cell type undergoes. Activated microglia demonstrate larger soma size, fewer process endpoints, shorter processes, and reductions in overall cell complexity as compared to non-activated ramified surveying microglia 29,40,54,65 . Activated astrocytes undergo hypertrophy and increased expression of the intermediate filament, GFAP 66,67 . Such activation-associated morphological changes of both microglia and astrocytes are seen in various brain regions following diffuse TBI 29,39,40,66,68,69 , however, the effects of Bup on such changes are not well understood. Both microglia and astrocytes express all three opioid receptors, suggesting that Bup could potentially play a role in modulation of microglial and/or astrocyte morphological changes associated with activation 10 . Our current studies found that Bup treatment was linked to a more activated morphological phenotype of microglia. Previous studies found that opioids given during development drastically reduce microglial process branching and hence the overall size and complexity of microglia; morphological changes indicative of microglial activation 9 . Other studies found that microglial activation is increased in a model of chronic morphine administration but can be reduced by the non-specific opioid receptor antagonist, Naloxone 70,71 . The effect of Bup on the immune system is not fully understood, but there is likely an interplay between its roles as a partial agonist at the MOR and antagonistic properties toward the other opioid receptors. Our findings also indicate potential regional specificity of Bup-associated microglial activation. When comparing the microglial morphology between the two regions in both saline and Bup-treated animals, cortical microglia were found to exhibited larger soma, reduced process network complexity, shorter maximum process length/cell, and decreased numbers of process endpoints per microglia than the thalamus, regardless of treatment group. Additionally, microglia in the cortex demonstrated morphological changes associated with activation (larger cell bodies and reduced process network complexities), whereas these Bup-associated microglial changes were not observed in the thalamus. Alternatively, Bup-treated rats demonstrated an increase in cell size, indicative of activation-linked hypertrophy, in the thalamus that was not discernable in the cortex of Bup-treated rats. Previous studies have found that astrocyte signaling is affected by buprenorphine. Treatment with Bup was associated with an upregulation of GFAP intensity 72 . Another study, however, found that Bup treatment reduced GFAP intensity in a model of morphine-induced dependence, demonstrating that astrocyte activation could be impacted by the interplay of multiple opioids 73 . Because Bup is a partial agonist of the MOR, at higher doses, Bup could induce an inhibition of the MOR that could impact microglial and astrocyte activation differentially 74 . The MOR is more highly expressed in the thalamus as compared to the lateral neocortex, which has low to moderate opioid receptor expression 75 . Therefore, the regional-specific differences between Bup effects on microglial and astrocyte morphologies could be linked to differences in the opioid receptor profiles between these two brain regions. A previous study found that Bup exposure during development had a significant impact on myelin protein expression. Specifically, it was found that a low dose of 0.3mg/kg Bup given to early postnatal rat pups resulted in significantly increased expression of all MBP isoforms 23 . However, when given at a higher dose of 1mg/kg, expression of MBP was delayed 74 . While our study utilized a dose of 1mg/kg Bup-SR-Lab, we found no change in overall MBP expression between the Bup treatment group and the saline treatment group. We did, however, observe regional differences between the cortex and, with higher expression of MBP in the cortex than in the thalamus (Figure 7F). This could be due to the adult age at which our study was conducted, in which developmental myelination has already occurred and delays in MBP expression upon injury and subsequent repair might be more subtle. The myelin protein, MBP, consists of four major isoforms with molecular weights of 21.5 kDa, 18.5 kDa, 17.2 kDa, and 14.0 kDa, produced via alternate splicing of the primary MBP transcript 41 . One study investigating the degradation of MBP following TBI reported that all four major isoforms of MBP were degraded within hours following injury and intact protein levels did not return to base levels for 3-5 days post-injury 76 . This study observed significant proteolysis of MBP in the cortex hours post-injury but found MBP breakdown in the hippocampus was more delayed with MBP breakdown not peaking until 48 h after TBI potentially due to differences in the compression-induced contusion force 76 . It is possible that in the current study, we observed higher levels of MBP expression in the cortex as compared to the thalamus due to such differences in biomechanical forces, however, in this study we were assessing MBP and not the breakdown of MBP. It is also possible that the observed difference in MBP expression in the cortex vs. the thalamus is reflective of the proportion of myelinated fibers within each region. Ultimately, it is likely that myelin pathology alterations could evolve over longer post-injury time-points following CFPI. Conclusions The findings of this initial study show that preclinical use of Bup-SR-Lab has little effect on acute pathology following diffuse brain injury. However, because only one acute time point was observed in this study, future studies are required to examine potential long-term differences in physiological and pathological outcomes following TBI. Additionally, as morphological aspects of both microglia and astrocytes were found to be changed by Bup-SR-Lab treatment, metanalytical studies assessing these parameters should be aware of the potential effects of post-injury Bup treatment on these outcome metrics. These subtle changes, however, would not preclude the use of Bup-SR-Lab for post-TBI pain management in primary acute-survival animal studies. Abbreviations TBI: Traumatic brain injury Bup-SR-Lab: buprenorphine-Slow-release-Lab CFPI: central fluid percussion injury ROI: region of interest MBP: myelin basic protein DAI: diffuse axonal injury FITBIR: Federal Interagency Traumatic Brain Injury Research Bup: buprenorphine H&E: hematoxylin and eosin Cat: catalogue SEM: standard error of the mean APP: amyloid precursor protein MOR: Mu opioid receptor KOR: Kappa opioid receptor DOR: delta opioid receptor CNS: central nervous system GFAP: glial fibrillary acidic protein Declarations Ethics approval Experiments were conducted in accordance with the Virginia Commonwealth University institutional ethical guidelines concerning the care and use of laboratory animals (Institutional Animal Care and Use Committee, Virginia Commonwealth University), which adhere to regulations including, but not limited to, those set forth in the “Guide for the Care and Use of Laboratory Animals: 8th Edition” (National Research Council). Consent for publication Not Applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by grant NINDS R01NS096143. Authors' contributions JR conducted the myelin pathology and MBP expression analyses and wrote the manuscript. PS carried out the histological assessments and wrote the paper. SL, BP and KG carried out the histological assessments. ADL developed the histological assessments, conceived, designed and coordinated the study and wrote the manuscript. Acknowledgements The authors would like to recognize Judy Williamson, Frances White and Susan Walker for invaluable technical assistance. This work was supported by NINDS grant 1R01NS096143. References Coronado, V. G. et al. Surveillance for traumatic brain injury-related deaths--United States, 1997-2007. MMWR. Surveill. Summ. 60 , 1–32 (2011). Ortiz-Prado, E. et al. A Nationwide Study of Incidence and Mortality Due to Traumatic Brain Injury in Ecuador (2004-2016). Neuroepidemiology 1–12 (2019) doi:10.1159/000502580. Bramlett, H. M. & Dietrich, W. D. Pathophysiology of cerebral ischemia and brain trauma: similarities and differences. J Cereb Blood Flow Metab 24 , 133–150 (2004). Home | FITBIR. (2019). National Research Council. GUIDE LABORATORY ANIMALS FOR THE CARE AND USE OF Eighth Edition Committee for the Update of the Guide for the Care and Use of Laboratory Animals Institute for Laboratory Animal Research Division on Earth and Life Studies . (2011). National Institutes of Health. Public Health Service Policy on Humane Care and Use of Laboratory Animals. Office Lab. Anim. Welf. 2002 (2013). Sun, J., Guo, W. & Du, X. Buprenorphine differentially affects M1- and M2-polarized macrophages from human umbilical cord blood. Eur. Cytokine Netw. 28 , 85–92 (2017). Corkrum, M., Rothwell, P. E., Thomas, M. J., Kofuji, P. & Araque, A. Opioid-Mediated Astrocyte–Neuron Signaling in the Nucleus Accumbens. Cells 8 , 586 (2019). Jantzie, L. L. et al. Prenatal opioid exposure: The next neonatal neuroinflammatory disease. Brain. Behav. Immun. 84 , 45–58 (2020). Murphy, A. et al. The Effects of Opioids on HIV Neuropathogenesis. Front. Immunol. 10 , (2019). Ilbäck, N.-G., Siller, M. & Stålhandske, T. Effects of buprenorphine on body temperature, locomotor activity and cardiovascular function when assessed by telemetric monitoring in rats. Lab. Anim. 42 , 149–160 (2008). Villiger, J. W. & Taylor, K. M. Buprenorphine: Characteristics of binding sites in the rat central nervous system. Life Sci. 29 , 2699–2708 (1981). Boas, R. A. & Villiger, J. W. CLINICAL ACTIONS OF FENTANYL AND BUPRENORPHINE The Significance of Receptor Binding . Br.J. Anaesth vol. 57 (1985). Greenwald, M. K., Comer, S. D. & Fiellin, D. A. Buprenorphine maintenance and mu-opioid receptor availability in the treatment of opioid use disorder: Implications for clinical use and policy. Drug Alcohol Depend. 144 , 1–11 (2014). Welsh, C. & Valadez-Meltzer, A. Buprenorphine: a (relatively) new treatment for opioid dependence. Psychiatry (Edgmont). 2 , 29–39 (2005). Chum, H. H. et al. Antinociceptive effects of sustained-release buprenorphine in a model of incisional pain in rats (Rattus norvegicus). J. Am. Assoc. Lab. Anim. Sci. 53 , 193–197 (2014). Falcon, E. et al. Antidepressant-like Effects of Buprenorphine are Mediated by Kappa Opioid Receptors. Neuropsychopharmacology 41 , 2344–2351 (2016). Kendall, L. V et al. Pharmacokinetics of sustained-release analgesics in mice. J. Am. Assoc. Lab. Anim. Sci. 53 , 478–484 (2014). Johnson, R. A. Voluntary running-wheel activity, arterial blood gases, and thermal antinociception in rats after 3 buprenorphine formulations. J. Am. Assoc. Lab. Anim. Sci. 55 , 306–311 (2016). Seymour, T. L. et al. Postoperative analgesia due to sustained-release buprenorphine, sustained-release meloxicam, and carprofen gel in a model of incision pain in rats (Rattus norvegicus). Jaalas 55 , 300–305 (2016). Foley, P. L., Liang, H. & Crichlow, A. R. Evaluation of a sustained-release formulation of buprenorphine for analgesia in rats. J. Am. Assoc. Lab. Anim. Sci. 50 , 198–204 (2011). DeClue, A. E. et al. Effects of opioids on phagocytic function, oxidative burst capacity, cytokine production and apoptosis in canine leukocytes. Vet. J. 200 , 270–275 (2014). Sanchez, E. S., Bigbee, J. W., Fobbs, W., Robinson, S. E. & Sato-Bigbee, C. Opioid addiction and pregnancy: Perinatal exposure to buprenorphine affects myelination in the developing brain. Glia 56 , 1017–1027 (2008). Lafrenaye, A. D., Krahe, T. E. & Povlishock, J. T. Moderately Elevated Intracranial Pressure after Diffuse Traumatic Brain Injury is Associated with Exacerbated Neuronal Pathology and Behavioral Morbidity in the Rat. J. Cereb. Blood Flow Metab. 34 , 1628–1636 (2014). Hernandez, M. L., Chatlos, T., Gorse, K. M. & Lafrenaye, A. D. Neuronal Membrane Disruption Occurs Late Following Diffuse Brain Trauma in Rats and Involves a Subpopulation of NeuN Negative Cortical Neurons. Front. Neurol. 10 , (2019). Dixon, C. E. et al. A fluid percussion model of experimental brain injury in the rat. J. Neurosurg. 67 , 110–119 (1987). Gorse, K. M. & Lafrenaye, A. D. The importance of inter-species variation in traumatic brain injury-induced alterations of microglial-axonal interactions. Front. Neurol. 9 , 778 (2018). Lafrenaye, A. D., McGinn, M. J. & Povlishock, J. T. Increased intracranial pressure after diffuse traumatic brain injury exacerbates neuronal somatic membrane poration but not axonal injury: Evidence for primary intracranial pressure-induced neuronal perturbation. J. Cereb. Blood Flow Metab. 32 , 1919–1932 (2012). Lafrenaye, A. D., Todani, M., Walker, S. A. & Povlishock, J. T. Microglia processes associate with diffusely injured axons following mild traumatic brain injury in the micro pig. J. Neuroinflammation 12 , 186 (2015). Simon, C. M., Sharif, S., Tan, R. P. & LaPlaca, M. C. Spinal cord contusion causes acute plasma membrane damage. J. Neurotrauma 26 , 563–574 (2009). Johnson, V. E., Stewart, W. & Smith, D. H. Axonal pathology in traumatic brain injury. Exp. Neurol. 246 , 35–43 (2013). Benson, C. Diffuse Axonal Injury. in Encyclopedia of the Neurological Sciences 998–999 (Elsevier, 2014). doi:10.1016/B978-0-12-385157-4.00326-2. Smith, D. H., Meaney, D. F. & Shull, W. H. Diffuse axonal injury in head trauma. J Head Trauma Rehabil 18 , 307–316 (2003). Geddes, D. M., LaPlaca, M. C. & Cargill, R. S. Susceptibility of hippocampal neurons to mechanically induced injury. Exp. Neurol. 184 , 420–427 (2003). LaPlaca, M. C. et al. Mechanoporation is a potential indicator of tissue strain and subsequent degeneration following experimental traumatic brain injury. Clin. Biomech. 64 , 2–13 (2019). Whalen, M. J. et al. Acute plasmalemma permeability and protracted clearance of injured cells after controlled cortical impact in mice. J. Cereb. Blood Flow Metab. 28 , 490–505 (2008). Farkas, O., Lifshitz, J. & Povlishock, J. T. Mechanoporation Induced by Diffuse Traumatic Brain Injury: An Irreversible or Reversible Response to Injury? J. Neurosci. 26 , 3130 LP – 3140 (2006). Prado, G. R. & LaPlaca, M. C. Neuronal Plasma Membrane Integrity is Transiently Disturbed by Traumatic Loading. Neurosci. Insights 15 , (2020). Loane, D. J., Kumar, A., Stoica, B. A., Cabatbat, R. & Faden, A. I. Progressive Neurodegeneration After Experimental Brain Trauma. J. Neuropathol. Exp. Neurol. 73 , 14–29 (2014). Byrnes, K. R., Loane, D. J., Stoica, B. a, Zhang, J. & Faden, A. I. Delayed mGluR5 activation limits neuroinflammation and neurodegeneration after traumatic brain injury. J. Neuroinflammation 9 , 43 (2012). Akiyama, K., Ichinose, S., Omori, A., Sakurai, Y. & Asou, H. Study of expression of myelin basic proteins (MBPs) in developing rat brain using a novel antibody reacting with four major isoforms of MBP. J. Neurosci. Res. 68 , 19–28 (2002). Santiago, J. M. et al. Molecular, anatomical, physiological, and behavioral studies of rats treated with buprenorphine after spinal cord injury. J. Neurotrauma 26 , 1783–1793 (2009). Dooley, S. B. et al. Pharmacokinetics and pharmacodynamics of buprenorphine and sustained-release buprenorphine after administration to adult alpacas. Am. J. Vet. Res. 78 , 321–329 (2017). Guarnieri, M. et al. Safety and efficacy of buprenorphine for analgesia in laboratory mice and rats. Lab Animal vol. 41 337–343 (2012). Dahan, A. et al. Comparison of the respiratory effects of intravenous buprenorphine and fentanyl in humans and rats. Br. J. Anaesth. 94 , 825–834 (2005). Dahan, A. et al. Buprenorphine induces ceiling in respiratory depression but not in analgesia. Br. J. Anaesth. 96 , 627–632 (2006). Cooper, C. S., Metcalf-Pate, K. A., Barat, C. E., Cook, J. A. & Scorpio, D. G. Comparison of side effects between buprenorphine and meloxicam used postoperatively in Dutch belted rabbits (Oryctolagus cuniculus). J. Am. Assoc. Lab. Anim. Sci. 48 , 279–285 (2009). Martin-Flores, M. et al. Effects of buprenorphine, methylnaltrexone, and their combination on gastrointestinal transit in healthy New Zealand white rabbits. J. Am. Assoc. Lab. Anim. Sci. 56 , 155–159 (2017). Carroll, F. I. & Carlezon, W. A. Development of κ opioid receptor antagonists. J. Med. Chem. 56 , 2178–2195 (2013). Elkader, A. & Sproule, B. Buprenorphine Clinical Pharmacokinetics in the Treatment of Opioid Dependence . Clin Pharmacokinet vol. 44 https://link-springer-com.proxy.library.vcu.edu/content/pdf/10.2165%2F00003088-200544070-00001.pdf (2005). Brown, S. M., Holtzman, M., Kim, T. & Kharasch, E. D. Buprenorphine Metabolites, Buprenorphine-3-glucuronide and Norbuprenorphine-3-glucuronide, Are Biologically Active . http://pubs.asahq.org/anesthesiology/article-pdf/115/6/1251/256341/0000542-201112000-00020.pdf (2011). Sabol Jones, M. K. & Ross, D. T. The partial μ opiate agonist buprenorphine protects a sub-population of thalamic reticular neurons following cardiac arrest in rats. Neurosci. Lett. 185 , 91–94 (1995). Lafrenaye, A. D., Krahe, T. E. & Povlishock, J. T. Moderately elevated intracranial pressure after diffuse traumatic brain injury is associated with exacerbated neuronal pathology and behavioral morbidity in the rat. J. Cereb. Blood Flow Metab. 1–9 (2014) doi:10.1038/jcbfm.2014.122. Thomas, T. C. et al. Does time heal all wounds? Experimental diffuse traumatic brain injury results in persisting histopathology in the thalamus. Behav. Brain Res. 340 , 137–146 (2018). Lafrenaye, A. D., McGinn, M. J. & Povlishock, J. T. Increased intracranial pressure after diffuse traumatic brain injury exacerbates neuronal somatic membrane poration but not axonal injury: evidence for primary intracranial pressure-induced neuronal perturbation. J Cereb Blood Flow Metab 32 , 1919–1932 (2012). Kelley, B. J., Lifshitz, J. & Povlishock, J. T. Neuroinflammatory responses after experimental diffuse traumatic brain injury. J. Neuropathol. Exp. Neurol. 66 , 989–1001 (2007). Witcher, K. G. et al. Traumatic brain injury-induced neuronal damage in the somatosensory cortex causes formation of rod-shaped microglia that promote astrogliosis and persistent neuroinflammation. Glia 66 , 2719–2736 (2018). Rowe, R. K. et al. Diffuse traumatic brain injury induces prolonged immune dysregulation and potentiates hyperalgesia following a peripheral immune challenge. Mol. Pain 12 , 1–12 (2016). Coughlin, J. M. et al. Neuroinflammation and brain atrophy in former NFL players: An in vivo multimodal imaging pilot study. Neurobiol. Dis. 74 , 58–65 (2015). Velázquez, A., Ortega, M., Rojas, S., González-Oliván, F. J. & Rodríguez-Baeza, A. Widespread microglial activation in patients deceased from traumatic brain injury. Brain Inj. 29 , 1126–1133 (2015). Ziebell, J. M. & Morganti-Kossmann, M. C. Involvement of Pro- and Anti-Inflammatory Cytokines and Chemokines in the Pathophysiology of Traumatic Brain Injury. Neurotherapeutics 7 , 22–30 (2010). Roy, S. et al. Do All Opioid Drugs Share the Same Immunomodulatory Properties? A Review From Animal and Human Studies. Front. Immunol. | www.frontiersin.org 10 , 2914 (2019). Félix, N. M. et al. Effects of buprenorphine in the adrenal, thyroid, and cytokine intra-operative responses in a rat model (Rattus norvegicus): a preliminary study. doi:10.22038/IJBMS.2017.8576. Muscat, S. M. et al. Post-operative cognitive dysfunction is made persistent with morphine treatment in aged rats. Neurobiol. Aging (2020) doi:10.1016/j.neurobiolaging.2020.11.008. Morrison, H. W. & Filosa, J. a. A quantitative spatiotemporal analysis of microglia morphology during ischemic stroke and reperfusion. J. Neuroinflammation 10 , 4 (2013). Clément, T. et al. Juvenile mild traumatic brain injury elicits distinct spatiotemporal astrocyte responses. Glia 68 , 528–542 (2020). Pekny, M. & Nilsson, M. Astrocyte activation and reactive gliosis. Glia 50 , 427–434 (2005). Hall, K. D. & Lifshitz, J. Diffuse traumatic brain injury initially attenuates and later expands activation of the rat somatosensory whisker circuit concomitant with neuroplastic responses. Brain Res. 1323 , 161–73 (2010). Lafrenaye, A. D. et al. Circulating GFAP and Iba-1 levels are associated with pathophysiological sequelae in the thalamus in a pig model of mild TBI. Sci. Rep. 10 , 13369 (2020). Li, Z., Jia, X., Peng, X. & Gao, F. The interaction between spinal pdgfrβ and μ opioid receptor in the activation of microglia in morphine-tolerant rats. J. Pain Res. 13 , 1803–1810 (2020). Carranza-Aguilar, C. J. et al. Morphine and Fentanyl Repeated Administration Induces Different Levels of NLRP3-Dependent Pyroptosis in the Dorsal Raphe Nucleus of Male Rats via Cell-Specific Activation of TLR4 and Opioid Receptors NLRP3 · Opioids · Glia · TLR4 · NF-κB · Serotonin. Cell. Mol. Neurobiol. doi:10.1007/s10571-020-00957-5. Gerhold, K. J., Drdla-Schutting, R., Honsek, S. D., Forsthuber, L. & Sandkühler, J. Pronociceptive and antinociceptive effects of buprenorphine in the spinal cord dorsal horn cover a dose range of four orders of magnitude. J. Neurosci. 35 , 9580–9594 (2015). Wu, F. X. et al. Dezocine Alleviates Morphine-Induced Dependence in Rats. Anesth. Analg. 128 , 1328–1335 (2019). Vestal-Laborde, A. A., Eschenroeder, A. C., Bigbee, J. W., Robinson, S. E. & Sato-Bigbee, C. The opioid system and brain development: Effects of methadone on the oligodendrocyte lineage and the early stages of myelination. Dev. Neurosci. 36 , 409–421 (2014). Le Merrer, J., Becker, J. A. J., Befort, K. & Kieffer, B. L. Reward processing by the opioid system in the brain. Physiological Reviews vol. 89 1379–1412 (2009). Ming, C. L. et al. Extensive degradation of myelin basic protein isoforms by calpain following traumatic brain injury. J. Neurochem. 98 , 700–712 (2006). Cite Share Download PDF Status: Published Journal Publication published 21 Apr, 2021 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 03 Feb, 2021 Reviews received at journal 22 Jan, 2021 Reviewers agreed at journal 11 Jan, 2021 Reviewers invited by journal 06 Jan, 2021 Editor assigned by journal 06 Jan, 2021 Editor invited by journal 06 Jan, 2021 Submission checks completed at journal 04 Jan, 2021 First submitted to journal 31 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-138979","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":7436231,"identity":"051b4ee7-ad93-48d4-b9d8-64a2704c2759","order_by":0,"name":"Jane Ryu","email":"","orcid":"","institution":"Virginia Commonwealth University Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jane","middleName":"","lastName":"Ryu","suffix":""},{"id":7436232,"identity":"3f667394-656b-4125-a352-007c7bffa024","order_by":1,"name":"Phillip Stone","email":"","orcid":"","institution":"Virginia Commonwealth University Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Phillip","middleName":"","lastName":"Stone","suffix":""},{"id":7436233,"identity":"79a5c3e4-d4fb-48ce-9108-8e2da78f4b8a","order_by":2,"name":"Sabrina Lee","email":"","orcid":"","institution":"Godwin High School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sabrina","middleName":"","lastName":"Lee","suffix":""},{"id":7436234,"identity":"aa3ba8cf-e4f4-4526-848f-fba9331a86e0","order_by":3,"name":"Brighton Payne","email":"","orcid":"","institution":"University of Mary Washington","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Brighton","middleName":"","lastName":"Payne","suffix":""},{"id":7436235,"identity":"ab498ea4-d5f7-44fc-bce9-563f33927264","order_by":4,"name":"Karen Gorse","email":"","orcid":"","institution":"Virginia Commonwealth University Medical Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Karen","middleName":"","lastName":"Gorse","suffix":""},{"id":7436236,"identity":"55669efa-0277-4771-9b57-afded0a8ccc9","order_by":5,"name":"Audrey Lafrenaye","email":"data:image/png;base64,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","orcid":"","institution":"Virginia Commonwealth University Medical Center","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Audrey","middleName":"","lastName":"Lafrenaye","suffix":""}],"badges":[],"createdAt":"2020-12-31 16:44:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-138979/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-138979/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-021-88030-z","type":"published","date":"2021-04-21T19:02:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":4796844,"identity":"45955c7b-a9a0-4034-bf77-1ee5ef8515bc","added_by":"auto","created_at":"2021-01-07 22:58:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":227220,"visible":true,"origin":"","legend":"Weight loss was reduced in animals treated with Bup-SR-Lab at 1-day post-injury. Physiological readouts in saline and buprenorphine (Bup) treated adult male rats following central fluid percussion injury (cFPI). A Blood oxygen saturation, B heart rate, and C Intracranial pressure were unchanged with Bup. D Weight loss 1d following cFPI was less substantial in cFPI rats treated with Bup compared to saline. Mean±SEM. *p\u003c0.05 compared to saline. ","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-138979/v1/efae617b1af417cd99f981f0.png"},{"id":4796643,"identity":"ef7e9b9a-2f02-439d-9875-a7d33972e7b1","added_by":"auto","created_at":"2021-01-07 22:52:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2062864,"visible":true,"origin":"","legend":"Cell damage was not altered by Bup treatment. Representative photomicrographs from animals treated with A\u0026C saline or B\u0026D buprenorphine following injury and labeled with hematoxylin and eosin (H\u0026E) to assess cell damage and death in A\u0026B cortical and C,D thalamic regions of interest 1d post-injury. Inset a is a positive control of damaged cells demonstrating eosinophilic cytoplasm and heterochromatic nuclei. E\u0026F Corresponding bar graphs depicting number of damaged cells per region of interest in cortex and thalamus. The number of damaged cells was consistent between saline and buprenorphine treated rats in both regions of interest, indicating no change in cell damage or death between treatment groups. Mean±SEM. Scale= 50μm.","description":"","filename":"Fig2copy.png","url":"https://assets-eu.researchsquare.com/files/rs-138979/v1/fc9e05266fff7217949465a8.png"},{"id":4796763,"identity":"627e1c3d-d425-42fb-bb48-45a9fa911b42","added_by":"auto","created_at":"2021-01-07 22:55:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3783394,"visible":true,"origin":"","legend":"Buprenorphine did not alter acute diffuse axonal injury in either the thalamus or cortex. Representative photomicrographs of A\u0026C saline or B\u0026D buprenorphine treated Sprague Dawley rats 1d following cFPI. Tissue samples of A\u0026B somatosensory neocortex and C\u0026D hemithalamus were labeled immunohistochemically with a primary antibody against amyloid precursor protein (APP) and a secondary biotinylated antibody followed by DAB reaction. Swellings were visualized and quantified to assess axonal injury in each brain region at 1d following cFPI and either saline or buprenorphine treatment. E\u0026F Corresponding bar graphs depicting the number of swellings per region of interest in the E cortex and F thalamus. The number of swellings was consistent between saline and buprenorphine treated animals, indicating that buprenorphine does not affect axonal injury in observed regions following TBI. n=6 rats/group; Mean±SEM. scale= 50μm.","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-138979/v1/e50d6f9dc1ab82846bacc942.png"},{"id":4796761,"identity":"a432dfcf-7e9d-4abb-9936-ed9c4df4f04e","added_by":"auto","created_at":"2021-01-07 22:55:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1879964,"visible":true,"origin":"","legend":"Acute neuronal membrane disruption was not altered in the cortex of Bup-treated animals. Representative photomicrographs of sub-acute neuronal membrane disruption in the lateral neocortex of rats treated with A saline or B buprenorphine. Prior to sacrifice at 1d post-injury rats were infused with a 10kDa cell-impermeable, biotinylated dextran. NeuN+ neurons are green and dextran is pseudocolored red. Brain sections were incubated with goat anti-biotin primary antibody followed by Alexa-568-conjugated donkey anti-goat secondary antibodies to visualized cells that contained dextrans for the assessment of neuronal membrane disruption (arrows) in the lateral neocortex layers V and VI. C Corresponding bar graph depicting the percentage of total neurons that showed disruption in saline and buprenorphine treated animals. The percentage of dextran+ neurons was consistent between groups, indicating that buprenorphine treatment did not affect the percentage of membrane disrupted neurons in the cortex. Mean±SEM.","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-138979/v1/c1e4ef33eeab659b0e59f0c4.png"},{"id":4796647,"identity":"3fc92ded-545c-44a1-aa1a-6d06adb04ea1","added_by":"auto","created_at":"2021-01-07 22:52:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2121019,"visible":true,"origin":"","legend":"Microglia demonstrate a more active morphology in the cortex, but not the thalamus, following Bup-SR-Lab treatment. Representative photomicrographs of A\u0026B cortex and B\u0026C thalamus from rats 1d post-cFPI and treated with A\u0026C saline or B\u0026D buprenorphine (Bup). The microglial calcium binding protein, Iba-1, is labeled in green. Bar graphs depicting microglial morphological characteristics indicative of activation; E number of process end points/cell, F average soma size, G average maximum process length and H the average complexity of the microglial process network/cell. Microglial morphologies were significantly different in the cortex compared to microglia found within the thalamus, regardless of treatment. Additionally, microglial somal size and process network complexity was altered in the cortex, but not the thalamus of Bup-treated rats, suggesting regional specificity in the effects of Bup. Treatment differences: *p\u003c0.05 compared to saline; regional differences: #p\u003c0.05 compared to cortex. Mean±SEM. Scale= 50μm.","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-138979/v1/3cd23d9e2c2fbbf3233fbea7.png"},{"id":4796649,"identity":"1f95edf4-1dbc-45ff-b086-737cc3e5194f","added_by":"auto","created_at":"2021-01-07 22:52:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2099291,"visible":true,"origin":"","legend":"Astrocytes are larger with Bup-treatment in the thalamus, but not in the cortex. Representative photomicrographs of the astrocytic glial fibrillary acidic protein (GFAP) in A\u0026B cortex and C\u0026D thalamus from rats 1d post-cFPI and treated with A\u0026C saline or B\u0026D buprenorphine (Bup). Bar graphs depicting astrocyte E cell number, F % of GFAP+ coverage/image, G average astrocyte cell size, and H astrocyte cell circularity. There were more, larger GFAP+ astrocytes covering a greater area in the thalamus compared to the cortex of saline treated rats 1d following cFPI. Thalamic astrocytes were larger with Bup treatment compared to those in saline treated rats, however cortical astrocyte size was not significantly altered by Bup treatment. Treatment differences: *p\u003c0.05 compared to saline; regional differences: #p\u003c0.05 compared to cortex. Mean±SEM. Scale= 50μm.","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-138979/v1/99063738a2bb190f47bdd24f.png"},{"id":4796764,"identity":"c69a6244-48fb-49e4-a2ea-afdc0b93093d","added_by":"auto","created_at":"2021-01-07 22:55:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1766460,"visible":true,"origin":"","legend":"Acute myelin debris and MBP expression was unchanged with Bup treatment but was different between cortex and thalamus. Representative photomicrographs of myelin integrity in A\u0026B cortex treated with saline or buprenorphine (Bup) and C\u0026D thalamus treated with saline or Bup, analyzed using ImageJ. Representative bands for (E) Western blot analysis of actin protein (band at ~40 kDa) and myelin basic protein (MBP; bands at ~15-20 kDa). (F) Bar graph depicting average overall MBP expression (gray bars), as well as expression of 21.5 kDa (red bars), 18.5 kDa (orange bars), 17.2 kDa (blue bars), and 14.0 kDa (green bars) MBP isoforms in the cortex and thalamus of saline and Bup treated animals. Overall MBP expression and expression of individual MBP isoforms were not significant in either saline or Bup treatment groups. There were, however, significant regional differences with higher MBP expression in the cortex than in the thalamus. (G) Bar graph depicting average number of MBP fibers and MBP debris of cortex and thalamus in saline and Bup treated animals. The number of myelinated fibers was consistent between groups, indicating neither region was affected by buprenorphine treatment. Treatment differences: regional differences: #p\u003c0.05 compared to cortex. Mean±SEM. Scale= 50μm.","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-138979/v1/617e82c8a702444aaff3258d.png"},{"id":13644011,"identity":"65f0f03a-5f81-421b-82c1-05c3ba69bd54","added_by":"auto","created_at":"2021-09-17 09:14:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5610467,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-138979/v1/45e1a035-69d4-4354-bbaf-e3062b485e8f.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eBuprenorphine Alters Microglia and Astrocytes Acutely Following Diffuse Traumatic Brain Injury.\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eTraumatic brain injury (TBI) is an increasingly common phenomenon, with almost 2 million reported cases occurring annually in the US alone\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Overall, TBIs contribute to nearly 30% of all injury-related deaths, and account for significant healthcare cost and adverse health effects. Brain injury-induced pathology can be subclassified into focal and diffuse changes. Focal pathology of TBI results from the impact of the brain, and results in relatively homogenous sequelae of injury. Diffuse pathology, on the other hand, can be widely distributed throughout the brain in \u0026ldquo;pockets\u0026rdquo; of injury, and involves heterogenous cellular responses, ranging from diffuse axonal injury (DAI) and neuronal membrane disruption to neuroinflammatory changes and myelin pathology, making it difficult to track in the human population. Therefore, animal models of TBI are utilized for the rigorous assessments of TBI-induced pathology. These experimental TBI studies are being brought into greater alignment modeling the common data elements utilized in clinical TBI studies by groups such as the Federal Interagency Traumatic Brain Injury Research (FITBIR) data sharing group\u003csup\u003e4\u003c/sup\u003e making metanalysis of experimental studies much more feasible.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs various models of experimental diffuse TBI require surgical intervention, guidelines suggest the use of analgesics\u003csup\u003e5,6\u003c/sup\u003e. The most commonly used analgesics in experimental studies are opioids. However, there are indications that opioids may alter neuronal survival and possibly inflammation\u003csup\u003e7\u0026ndash;11\u003c/sup\u003e, and the effects of opioids on various other pathologies have not been rigorously tested. Because effects of analgesics on neuropathology and physiology are poorly described, it is unclear if their use confounds data. This uncertainty regarding possible secondary effects of opioid administration post-injury has led to a debate regarding the use of analgesics following TBI.\u003c/p\u003e\n\u003cp\u003eBuprenorphine (Bup) is a semi-synthetic opioid derived from thebaine, one of six naturally occurring alkaloids of the opium poppy\u003csup\u003e10,12\u003c/sup\u003e. It is a partial agonist of the Mu receptor and an antagonist of the Kappa and Delta opioid receptors\u003csup\u003e12,13\u003c/sup\u003e. Due to these binding properties, Bup is commonly used in the treatment of opioid use disorder, the context in which the drug is most often studied\u003csup\u003e14,15\u003c/sup\u003e. Bup is also the most commonly used analgesic in pre-clinical animal models owing to the effectiveness of the sustained release formulation of Bup, Bup-SR-lab, in pain reduction over multiple days following a single subcutaneous administration\u003csup\u003e16\u0026ndash;21\u003c/sup\u003e. While there are indications that Bup could affect cellular pathology\u003csup\u003e7,22,23\u003c/sup\u003e, the specific effects of Bup-SR-Lab on neuronal/glial pathology, neuroinflammation, cell damage, and physiology following brain injury are still unclear. Therefore, this study sought to determine the effects of Bup-SR-Lab on various acute diffuse pathologies precipitated by TBI. Specifically, physiological changes, neuronal membrane disruption, axonal injury, microglial and astrocyte alterations, cytokine expression and myelin changes were assessed at 1 day following diffuse central fluid percussion injury (CFPI) in adult male Sprague-Dawley rats treated subcutaneously with 1mg/kg Bup-SR-Lab or saline at 15min post-injury.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were conducted in accordance with ARIVE guidelines and the Virginia Commonwealth University institutional ethical guidelines concerning the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Virginia Commonwealth University, which adhere to regulations including, but not limited to, those set forth in the \u0026ldquo;Guide for the Care and Use of Laboratory Animals: 8th Edition\u0026rdquo; (National Research Council).\u0026nbsp;Overall, 12 adult (12 to 16-week-old; n=6/group) male Sprague-Dawley rats were used for this study. Animals were housed in individual cages on a 12-hour light-dark cycle with free access to food and water and full veterinary oversight.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurgical Preparation, Injury Induction, and Drug Administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnesthesia was induced with 4% isoflurane in 30% O\u003csub\u003e2\u003c/sub\u003e/70% room air. Animals were then intubated and ventilated with 1.5%-2.5% isoflurane in 30% O\u0026shy;\u003csub\u003e2\u003c/sub\u003e and 70% room air throughout the duration of the surgery, injury, and physiologic monitoring. Body temperature was maintained at 37\u003csup\u003eo\u003c/sup\u003eC with a rectal thermometer connected to a feedback-controlled heating pad (Harvard Apparatus, Holliston, MA, USA). All animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). A midline incision was made followed by a 4.8 mm diameter circular craniectomy, which was positioned along the sagittal suture midway between bregma and lambda. The dura was left intact. A 2mm diameter burr hole was also drilled into the left parietal bone overlaying the left lateral ventricle (0.8 mm posterior, 1.3 mm lateral, and 2.5 to 3 mm ventral relative to bregma) through which a 25-gauge needle, connected to a pressure transducer and a micro infusion pump 11 Elite syringe pump (Harvard Apparatus) via PE50 tubing, was placed into the left ventricle. Appropriate placement of the infusion pump into the lateral ventricle was verified via a 2.3\u0026mu;l/min infusion of sterile saline within the closed fluid pressure system during needle placement\u003csup\u003e24,25\u003c/sup\u003e. The needle was held in the lateral ventricle for at least 5 minutes to record preinjury ICP; then the needle was slowly removed. Bone wax was used to seal the burr hole used for the ICP measurements before preparation for central fluid percussion injury (CFPI). The procedures used to induce CFPI were consistent with those described previously\u003csup\u003e24\u0026ndash;27\u003c/sup\u003e. Briefly, a Luer-Loc syringe hub was affixed to the craniotomy site with dental acrylic (methyl methacrylate; Hygenic, Akron, OH, USA) that was applied around the hub, including the area overlying the sealed burr hole and allowed to harden. Animals were removed from the stereotaxic frame and placed on a raised platform for connection to the fluid percussion device, maintaining an unbroken fluid-filled system from the intact dura through the cylinder, via a Leur-Loc adaptor. During injury the investigator supported the animal\u0026rsquo;s body on the platform but did not hold the head allowing the Leur-Loc mechanism to maintain connection between the injury hub and fluid percussion device. To induce a mild-moderate CFPI a pendulum was released onto the fluid-filled cylinder of the FPI device, producing a pressure pulse of 2.05\u0026plusmn;0.10 atmospheres for ~22.5msec (table 1), which was transduced through the intact dura to the CSF. The pressure pulse was measured by a transducer affixed to the injury device and displayed on an oscilloscope (Tektronix, Beaverton, OR, USA). Immediately after the injury, animals were reconnected to the ventilator and physiologic monitoring devices. The hub, dental acrylic, and bone wax were removed \u003cem\u003een bloc\u003c/em\u003e and Gelfoam was placed over the craniectomy/injury site. The animal was then replaced in the stereotaxic frame, and the ICP probe was reinserted into the lateral ventricle, as described above, for postinjury ICP monitoring. Immediate post-injury physiology was recorded for 15min after CFPI followed by subcutaneous administration of either 1mg/kg Bup SR-Lab or saline. The surgeon randomly selected a pre-filled blinded syringe that was administered by another investigator to avoid inadvertent unblinding of the surgeon due to the difference in viscosity of the solutions that might introduce bias that could influence animal care. One hour following injury the scalp was sutured and treated with lidocaine and triple-antibiotic ointment. Rats were then allowed to recover and were returned to clean home cages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiologic Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeart rate, respiratory rate, and hemoglobin oxygen saturation were monitored via a hindpaw pulse oximetry sensor (STARR Life Sciences, Oakmont, PA, USA) for the duration of anesthesia, except during the induction of injury. Intracranial pressure (ICP) was measured intraventricularly, as described above. All physiologic measurements were recorded using a PowerLab System (AD Instruments, Colorado Springs, CO, USA). All animals maintained systemic physiological homeostasis throughout the experiment (i.e., heart rate\u0026gt;200 beats per min and oxygenation\u0026gt;90%; Table 1; Figure 1). Changes in ICP following CFPI were noted in both groups, particularly at 1d post-injury (Table 1; Figure 1C). Recovery time following CFPI (the time from withdrawal of inhaled anesthetic to first movement) and weight loss (percent reduction in animal weight from pre-injury to 1d post-injury) were also assessed (Table 1; Figure 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTracer Infusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 1d post-injury rats were anesthetized with 4% isoflurane in 30% O\u003csub\u003e2\u003c/sub\u003e/70% room air followed by maintenance dose of 2% isoflurane in 30% O\u003csub\u003e2\u003c/sub\u003e/70% room air via a nose cone. Animals were secured into a stereotaxic device and the incision from the previous day was reopened by removing the sutures. The ICP needle was filled with 0.7mg/17\u0026mu;l 10kDa biotinylated dextran and placed into the left lateral ventricle with continuous ICP monitoring, as described above. The ICP at 1d post-CFPI was measured for 15min following needle placement then the dextran was infused at a rate of 1.3\u0026mu;l/min with continuous ICP monitoring. The tracer was allowed to diffuse for 2h before animals underwent transcardial perfusion, as described below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue Processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt 1d post-CFPI 2hr following tracer infusion, anesthetized rats were overdosed with Euthasol euthanasia-III solution (Henry Schein, Dublin, OH, USA) followed by transcardial perfusion with cold 0.9% saline. As was described previously\u003csup\u003e25\u003c/sup\u003e, both fresh and fixed brain tissue was collected from each animal. A tissue core of the right lateral neocortex and thalamus/midbrain was taken for molecular assessments prior to transcardial fixation with 4% paraformaldehyde/0.2% glutaraldehyde in Millonig\u0026rsquo;s buffer (136 mmol/L sodium phosphate monobasic/109 mmol/L sodium hydroxide) for immunohistochemical analysis of the left side of the brain. After transcardial perfusion, the left side of the brain was removed and postfixed for \u0026gt;72h. Postfixed brains were sectioned coronally in 0.1 mmol/L phosphate buffer with a vibratome (Leica, Banockburn, IL, USA) at a thickness of 40 \u003cem\u003e\u0026mu;\u003c/em\u003em from bregma to \u0026sim;4.0 mm posterior to bregma. Sections were collected serially in 12-well plates and stored in MIllonig\u0026rsquo;s buffer at 4\u003csup\u003eo\u003c/sup\u003eC. All quantitative analyses were performed at least 1 mm posterior to the needle track used for ICP monitoring. The well from which sections would be taken for analysis was selected via a random number generator (1-12) and the first 4 sections with visible hippocampus were taken representing serial sections throughout the rostral-caudal extent (1.8 mm\u0026plusmn;0.2 mm to 3.8 mm\u0026plusmn;0.2 mm posterior to bregma, each 480\u0026mu;m apart). Histologic analyses were performed on the left lateral somatosensory cortex restricted to layers V and VI extending from the area lateral to CA1 to the area lateral to CA3 of the hippocampus and entire left hemi-thalamus extending from the midline and dorsal surface of the thalamus to the reticular nucleus and zona inserta of the thalamus laterally and ventrally.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Cell Damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the numbers of damaged cells in the cortex and thalamus of rats following injury and vehicle or Bup treatment, four sequential, randomly selected sections per animal were stained with hematoxylin and eosin (H\u0026amp;E) and assessed as described previously\u003csup\u003e24,25,28\u003c/sup\u003e. Briefly, tissue was mounted on gelatin-coated slides before dehydration and rehydration. Rehydrated tissue was incubated in Gills hematoxylin (Leica Biosystems, Buffalo Grove, IL, USA) followed by bluing agent (Leica Biosystems) and three dips in 0.25% eosin Y/0.005% acetic acid/95% ethanol before sections were cleared through increasing concentrations of ethanol and cover-slipped with Permount (Thermo Fisher Scientific, Waltham, MA, USA). Sections were visualized using a Nikon Eclipse 800 microscope. Assessments were done on the left side of the cortex and thalamus for each section. The number of damaged neurons, delineated by eosinophilic cytoplasm and condensed nuclei, in the entire left lateral neocortex and thalamus was counted by two independent investigators blinded to the animal group and averaged for each animal and each group. Data is reported as the number of damaged cells/region of interest (ROI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Axonal Injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify axonal injury, immunohistochemistry targeting amyloid precursor protein (APP) was performed. Sections were immunolabeled, as previously described\u003csup\u003e24,29\u003c/sup\u003e. Tissue was blocked and permeabilized in 5% normal goat serum and 1.5% triton followed by overnight incubation with a primary rabbit antibody against the C terminus of \u003cem\u003e\u0026beta;\u003c/em\u003e-APP (Cat. #51-2700, 1:700, Life Technologies). Secondary antibody, biotinylated goat anti-rabbit IgG (Cat. #BA-1000, 1:1000, Vector Laboratories, Burlingame, CA, USA) was then incubated for 2h at room temperature. The sections were subsequently incubated in avidin biotinylated enzyme complex using the Vectastain ABC kit (Vector Laboratories) followed by visualization with 0.05% diaminobenzidine/0.01% H\u003csub\u003e2\u003c/sub\u003e0\u003csub\u003e2\u0026shy;\u003c/sub\u003e/0.3% imidazole/phosphate-buffered saline. The tissue was mounted, dehydrated, and cover-slipped. Visualization of APP-labeled axonal swellings was performed using a Nikon Eclipse 800 microscope (Nikon, Tokyo, Japan) equipped with an Olympus DP71 camera (Olympus, Center Valley, PA, USA). The total number of APP+ axonal swellings in the entire region of interest (the left lateral neocortex layers V and VI or the left thalamus) for each section was counted by eye by an investigator blinded to animal group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify microglia, astrocytes, and myelin, fluorescent immunohistochemistry against the calcium binding protein, Iba-1 (microglia), glial fibrillary acidic protein, GFAP (astrocytes), or myelin basic protein, MBP (myelin) was done. Briefly, 40mm thick coronal sections were blocked and permeabilized in 1.5% triton and 5% normal goat serum followed by overnight incubation with primary antibody rabbit anti-Iba-1 (Cat. #019-19741, 1:1000, Wako; Osaka, Japan), mouse anti-GFAP (Cat.#MAB3402, 1:1000; Millipore Burlington, MA, USA) or mouse anti-myelin basic protein (Cat #808401; 1:1000; BioLegend, San Diego, CA, USA) overnight followed by incubation with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Cat.# A11034, 1:700; ThermoFisher Scientific) or Alexa Fluor 568-conjugated goat anti-mouse secondary antibody (Cat.# A-11031, 1:700; ThermoFisher Scientific). Tissue was mounted using Vectashield hardset mounting medium with Dapi (Cat.#H-1500; Vector Laboratories). Immunolabeling for all tissue was done at the same time to reduce run-to-run variability. All image acquisition settings were held consistent between groups for each region of interest (left lateral neocortex layers V and VI or the left thalamus) and imaging was done by an investigator blinded to animal group. Dapi nuclear labeling was used to verify focus and restriction within the regions of interest prior to image acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Neuronal Membrane Disruption \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsistent with previous studies, we assessed the potential for neuronal membrane disruption via the utilization of tagged 10 kDa dextran\u003csup\u003e24,25,30\u003c/sup\u003e, which are impermeable to cells with intact membranes. Cells containing dextran, therefore, indicate membrane disruption. Immunolabeling for all tissue was done at the same time to reduce run-to-run variability. Sections were blocked with 5% normal goat serum, permeabilized with 1.5% triton, and immunolabeled with primary antibodies mouse anti-NeuN (Cat. #MAB377, 1:500, Millipore), to identify neurons, and goat anti-biotin (Cat #31852, 1:2,000, ThermoFisher Scientific; Waltham, MA, USA) to identify dextran. Secondary antibodies Alexa-fluor 568-conjugated donkey anti-goat IgG (Cat. #A11057, 1:700, ThermoFisher Scientific) and Alexa-fluor 488-conjugated goat anti-mouse IgG (Cat. #A11001, 1:700, ThermoFisher Scientific) were then incubated and the tissue was mounted with Vectashield hardset mounting medium with DAPI (Cat.#H-1500; Vector Laboratories). Sections were imaged by confocal microscopy using a Zeiss LSM 700 System (Carol Zeiss, Oberkochen, Germany). Confocal images of the left neocortex layers V and VI were taken at x 40 magnification in a systematically random fashion by an investigator blinded to animal group using DAPI labeling to verify focus and NeuN label to verify location within the region of interest. Image acquisition settings were held constant for comparable regions for all groups analyzed. Analysis of NeuN\u003csup\u003e+\u003c/sup\u003e neurons containing the cell-impermeable dextran was performed by an investigator blinded to animal group using the ImageJ colocalization finder plugin and traditional cell counting. Dextran containing neurons were quantified for each image and averaged for each animal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Microglial Activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour sections/animal labeled with Iba-1 were imaged using an Olympus DP71 camera (Olympus, Center Valley, PA, USA) and were analyzed using FIJI/ImageJ as follows. All cells in which both the cell body and process network were in focus were marked in each image and a subset (n=5/image) were randomly selected using a random number generator for further morphological analysis. Each randomly selected cell was individually analyzed for process number, number of branch end/terminal points, and maximum process segment length using the skeleton analysis tool in ImageJ. A complexity index was also calculated for each microglia using the formula, complexity index= number of processes/number of end points, with a lower number indicating reduced process complexity. The soma of each cells was also circumscribed to assess the perimeter of the cell body through ImageJ particle analysis. All data was recorded by an investigator blinded to animal group and averaged for each image. Individual microglia were considered ns.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Astrocyte Activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree randomly selected images in a randomly selected section were taken at 20x magnification under consistent microscope settings for each region of interest (left lateral neocortex layers V and VI or the left thalamus) using a Keyence BZ-X800 microscope with section scanning on to reduce background (Keyence Corporation of America, Itasca, IL, USA). Images were processed with background subtraction and automatic thresholding to generate masks of GFAP+ astrocytes. All cells within the mask were added to the Region of Interest Manager in FIJI/ImageJ. Measurements of the number of astrocytes/image, cellular area and circularity of individual astrocytes and the percent of GFAP+ astrocyte coverage/image were assessed. All data was analyzed by an investigator blinded to animal group and averaged for each image and each animal. Individual animals were considered ns.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssessment of Myelin Integrity \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour sections per animal with 6 images/section for the cortex or 4 images/section for the hemi-thalamus were imaged at 40X magnification in a systematically random fashion for each ROI using a Keyence BZ-X800 microscope with section scanning on to reduce background (Keyence Corporation of America). All images were captures and analyzed by an investigator blinded to animal group using the DAPI label to verify focus and location within the ROI. Image acquisition settings were held constant for comparable regions (cortex or thalamus) for all groups analyzed. Analysis of intact myelin fibers and myelin debris was performed using the Analyze particle plugin in FIJI/ImageJ (National Institutes of Health) with size and circularity parameters for object differentiation. The parameters used to determine myelin debris were circularity=0.3-1.0 and particle size=0.5-10 \u0026micro;m\u003csup\u003e2\u003c/sup\u003e. To assess myelin fibers the analysis settings were as follows, circularity= 0.0-0.1 and particle size= 25-infinity \u0026micro;m\u003csup\u003e2\u003c/sup\u003e. The average total area covered by intact myelin fibers or myelin debris was quantified for each image and averaged for each animal. Individual animals were considered ns.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of Protein Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissue from the right lateral neocortex and thalamus was homogenized in NP40 Buffer (150mM NaCl, 50 mM Tris pH 8.0, 1% Triton-X) and protease inhibitor cocktail (AEBSF 10.4mM, Aprotinin 8\u0026mu;M, Bestatin 400\u0026mu;M, E-64 140\u0026mu;M, Leupeptin 8\u0026mu;M, Pepstatin A 150\u0026mu;M, Cat#: P8340, Sigma, Saint Louis, MO, USA). Protein concentration was determined using a bicinchoninic acid assay in accordance with manufacturer\u0026rsquo;s instructions (Cat#23225; ThermoFisher) and quantified on a PHERAstar Spectrophotometer (BMG Labtech, Cary, NC, USA).\u003c/p\u003e\n\u003cp\u003eFor assessment of cytokine expression, cortical or thalamic protein homogenates were sent to Quansys Bioscience (Logan, UT, USA) for cytokine analysis of rat IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-10, IL-12, IFNy, and TNFa. Three replicates were run for each sample and the means of the replicates were used for each animal. Cytokine concentration (in pg) was normalized to total protein concentrations (in mg) for each sample. Samples in which there was no detectable amount of cytokine were set to 0pg/mg for analysis (IL-1b Thalamus saline n=3, Thalamus Bup n=1; IL-12 Thalamus saline n=4, Thalamus Bup n=4) after verifying acceptable (\u0026gt;0.5mg/ml) total protein concentrations.\u003c/p\u003e\n\u003cp\u003eTo analyze myelin basic protein (MBP) expression, Western blotting was performed. Protein (15 ug) was boiled for 10 min in 2x Laemelli loading buffer and run at 200 volts for 30 min on Mini-PROTEAN TGX Stain-free 4-20% precast polyacrylamide gels (Cat #4568096; BioRad, Hercules, CA, USA). Protein was transferred onto 0.2 um PVDF membranes using a Transblot Turbo transfer system (Bio-Rad) under the low molecular weight manufacturer settings (2.5 Amps, 25 Volts for 5 min). Western blotting was done on an iBind flex apparatus (Invitrogen) using primary antibodies rat anti-myelin basic protein (1:1000, Cat #MAB386; Millipore Sigma) and mouse anti-actin (1:4000, Cat #66009-1-Ig; Proteintech; Rosemont, IL, USA) followed by anti-rat-HRP secondary antibody (1:5000; Cat#112-035-003; Jackson Laboratories, West Grove, PA) and anti-mouse-HRP secondary antibody (1:5000, Cat #115-035-003; Jackson Laboratories; West Grove, PA). Chemiluminescent images were taken on a ChemiDoc imaging system (BioRad). Densitometric analysis was done in ImageJ (National Institutes of Health) for actin and total MBP expression, as well as individual MBP isoform expression. Total MBP expression was measured by taking the densities of each individual MBP isoform band and adding them to get the total sum. This method reduced biasing of the analysis by the variable degrees of white space between the MBP isoform bands in each run. MBP was then normalized to actin and to sham controls. All western blots were run in triplicates on three separate gels to reduce run-to-run variability potentially biasing the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA Shapiro-Wilk test for normality of the data was done prior to utilizing non-parametric statistics for data that was not normally distributed. The number of animals to be assessed for each group was determined by power analysis using previous data, an alpha=0.05 and a power of 80%. One-way or two-way ANOVA were done with Bonferroni post-hoc corrections for multiple comparisons. Statistical significance was set to a p value \u0026lt;0.05. Data are presented as mean\u0026plusmn; standard error of the mean (SEM) unless otherwise indicated.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePost-injury weight loss was decreased with Bup-SR-Lab treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs has been well characterized previously, CFPI (2.05\u0026plusmn;0.1 atmospheres) did not generate gross tissue pathology\u003csup\u003e24,25,27,28\u003c/sup\u003e. While there was apparent sub-arachnoid bleeding at 1d post-injury, there was no apparent damage to the underlying cortex. Neither saline nor Bup treated animals demonstrated indications of contusion, hematoma formation, or overt cortical compression.\u003c/p\u003e\n\u003cp\u003eBody temperature was maintained at 37\u0026deg;C with a feedback loop thermoregulatory system connected to a rectal thermometer to avoid potential confounds of hypothermia in either group. As drug treatment was both randomized and blinded there was little possibility to fully match injury metrics between treatment groups, however, there was no difference in pre-injury weight (One-way ANOVA F\u003csub\u003e1,12\u003c/sub\u003e=0.73, p=0.411), injury intensity (One-way ANOVA F\u003csub\u003e1,12\u003c/sub\u003e=0.68, p=0.427), or injury duration (One-way ANOVA F\u003csub\u003e1,12\u003c/sub\u003e=0.013, p=0.913) between groups (Table 1). While recovery time (time from withdrawal from anesthesia to first sporadic movements) was higher in the Bup treated group on average, there was substantial variability in this group and ultimately this difference was not significant (Table 1; One-way ANOVA F\u003csub\u003e1,12\u003c/sub\u003e=2.82, p=0.124).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Physiological readouts in buprenorphine and saline treated animals prior to and following cFPI. g= grams, atm=atmospheric pressure, BPM=beats per minute, \u003cstrong\u003eD\u003c/strong\u003eICP 10min-1d=change in intracranial pressure from 10min post-injury to 1d post-injury. Data presented as mean (standard deviation).\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e\u003cstrong\u003eSaline\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e\u003cstrong\u003eBup\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003ePre-injury Weight (g)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e509 (87)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e545 (61)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003eInjury Intensity (atm)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e2.05 (0.08)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e2.01 (0.10)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003eInjury duration (msec)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e22.34 (0.64)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e22.37(0.21)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003eRecovery Time (min)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e19.6 (7.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e79.9 (78.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003eD\u003c/strong\u003e\u003cstrong\u003e ICP 10min-1d\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"90\"\u003e\n\u003cp\u003e8.51 (6.74)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"97\"\u003e\n\u003cp\u003e7.51 (4.04)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr /\u003eTo evaluate the potential effects of Bup on physiology, blood oxygen saturation, heart rate, and intracranial pressure were assessed prior to injury and at 1h and 1d following CFPI. Animal weight was also assessed prior to and 1d following CFPI to explore potential effects of Bup on post-injury weight loss. There was no discernable difference in blood oxygenation or heart rate between saline and Bup treated animals at any time point assessed (Figure 1 A\u0026amp;B). Intracranial pressure was also comparable between treatment groups at all time points measured (Figure 1C; Two-Way ANOVA F\u003csub\u003e1,22\u003c/sub\u003e=2.37; p=0.138). A significant increase in intracranial pressure of about 8mmHg from 1h to 1d post-injury was, however, noted in both treatment groups (Two-Way ANOVA F\u003csub\u003e1,22\u003c/sub\u003e=5.50; p=0.028). This change in intracranial pressure was also consistent between saline and Bup treated groups (Table 1). Weight loss, however, was significantly reduced in the Bup treated group (3.36\u0026plusmn;0.72% loss from pre-injury weight) as compared to the saline treated group (6.53\u0026plusmn;0.78% loss from pre-injury weight; One-way ANOVA F\u003csub\u003e1,11\u003c/sub\u003e=8.81; p=0.013). This difference in weight loss was not correlated to pre-injury weight (spearman Rho=0.14, p=0.648).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment with Bup-SR-Lab did not alter neuronal somatic or axonal injuries\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs CFPI in rats produces significant and various cellular pathology in the lateral neocortex and thalamic domains\u003csup\u003e24,27\u003c/sup\u003e, these regions of interest were the focus of all subsequent pathological assessments. Overt acute cellular damage/death was assessed using H\u0026amp;E staining. Any cell that demonstrated a heterochromatic nucleus and eosinophilic cytoplasm was considered to be damaged and/or undergoing early stages of cell death\u003csup\u003e25,27,28\u003c/sup\u003e (Figure 2a). There were very few damaged/dead cells in either the cortex (Figure 2 A-C; One-way ANOVA, F\u003csub\u003e1,10\u003c/sub\u003e=0.51, p=0.493) or the thalamus (Figure 2 D-F; One-way ANOVA F\u003csub\u003e1,10\u003c/sub\u003e=3.18, p=0.105) of either saline or Bup treated animals and no significant difference between groups was detected for either ROI.\u003c/p\u003e\n\u003cp\u003eDiffuse axonal injury (DAI) is a hallmark of mild and/or diffuse TBI and is the leading pathological indicator of injury magnitude following a brain injury\u003csup\u003e31\u0026ndash;33\u003c/sup\u003e. Therefore, the total number of amyloid precursor protein positive (APP+) axonal swellings, indicative of DAI-mediated axonal transport dysfunction, was assessed in both the lateral cortex and the thalamus 1d following CFPI and either saline or Bup treatment. While notable DAI was visible in both saline treated (cortex=17.10\u0026plusmn;3.39, Thalamus=89.33\u0026plusmn;35.50 APP+ swelling/ROI) and Bup treated (cortex=17.15\u0026plusmn;3.03, Thalamus=82.71\u0026plusmn;16.79 APP+ swelling/ROI) animals, the degree of DAI was indistinguishable between the two groups in either the cortex (Figure 3A-C; F\u003csub\u003e1,10\u003c/sub\u003e=1.4x10\u003csup\u003e-4\u003c/sup\u003e, p=0.990) or the thalamus (Figure 3D-F; One-way ANOVA F\u003csub\u003e1,10\u003c/sub\u003e=0.028, p=0.869).\u003c/p\u003e\n\u003cp\u003eFinally, as somatic neuronal damage, in the form of neuronal membrane disruption, is present acutely following both diffuse and focal TBI, and as this pathology is exacerbated by secondary insults, we investigated the potential effects of Bup on neuronal membrane disruption\u003csup\u003e24,25,28,34\u0026ndash;36\u003c/sup\u003e. As we and others have demonstrated previously, tagged 10kDa dextran, which is normally excluded from intact membranes, are a reliable way to evaluate neuronal plasmalemmal disruptions\u003csup\u003e24,25,28,35,37,38\u003c/sup\u003e. However, this method only allows assessment of the cortex, due to the variability of dextran diffusion within the thalamic domain. As found with axonal injury, there was no difference between saline treated and Bup treated groups in regard to the percent of neurons demonstrating membrane disruption 1d following CFPI (Figure 4; One-way ANOVA F\u003csub\u003e1,10\u003c/sub\u003e=0.192, p=0.671).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBuprenorphine treatment alters microglia and astrocytes in a region-specific manor acutely following diffuse TBI. \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the potential effects of Bup on acute inflammatory responses in the brain cytokine protein levels were assessed for each ROI in saline and Bup treated animals at 1d post-CFPI. Levels of IL-1a, IL-2, IL-6, IFNy, and TNFa were consistently below detectable limits for nearly all samples tested regardless of treatment group. Concentration of IL-1b, IL-4, IL-10, and IL-12, however, were measurable for the majority of samples tested. There did appear to be a regional difference in expression of IL-1b (Two-way ANOVA; Region F\u003csub\u003e1,16\u003c/sub\u003e=22.53, p=2.19x10\u003csup\u003e-4\u003c/sup\u003e), IL-4 (Two-way ANOVA; Region F\u003csub\u003e1,16\u003c/sub\u003e=38.53, p=1.3x10\u003csup\u003e-5\u003c/sup\u003e), and IL-10 (Two-way ANOVA; Region F\u003csub\u003e1,16\u003c/sub\u003e=35.75 p=1.9x10\u003csup\u003e-5\u003c/sup\u003e) with consistently more cytokine expression in the cortex as compared to the thalamus (Table 2). However, there was no discernable treatment effect in either region for any of the cytokines evaluated (Two-way ANOVA; Treatment IL-1b F\u003csub\u003e1,16\u003c/sub\u003e=0.08 p=0.787; IL-4 F\u003csub\u003e1,16\u003c/sub\u003e=0.63 p=0.439; IL-10 F\u003csub\u003e1,16\u003c/sub\u003e=0.312 p=0.584; IL-12 F\u003csub\u003e1,16\u003c/sub\u003e=0.619 p=0.443).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Cytokine concentrations in cortex and thalamus of saline and buprenorphine treated animals at 1d following CFPI. Data presented as mean (SD) pg of cytokine per mg of total protein. Regional differences: # p\u0026lt;0.05 compared to cortex.\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"249\"\u003e\n\u003cp\u003e\u003cstrong\u003eSaline\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"249\"\u003e\n\u003cp\u003e\u003cstrong\u003eBuprenorphine\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003eCortex\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003eThalamus\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003eCortex\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e\u003cstrong\u003eThalamus\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003eIL-1b pg/mg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e14.40 (4.44)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e0.36 (0.50) #\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e14.40 (11.45)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e2.29 (2.29) #\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003eIL-4\u0026nbsp; pg/mg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e12.78 (6.23)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e1.13 (1.15) #\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e12.97 (2.51)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e3.63 (3.29) #\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003eIL-10\u0026nbsp; pg/mg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e17.12 (7.71)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e4.36 (2.77) #\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e18.69 (3.79)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e5.23 (3.83) #\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"114\"\u003e\n\u003cp\u003e\u003cstrong\u003eIL-12\u0026nbsp; pg/mg\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e36.93 (23.04)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e19.64 (43.93)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e33.41 (28.87)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"125\"\u003e\n\u003cp\u003e2.84 (6.35)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTo further investigate the potential that more subtle changes in neuroinflammation were occurring acutely following Bup treatment, microglia and astrocyte morphologies were analyzed. Alterations in microglial morphology indicative of an altered activation state (larger soma size, fewer process endpoints, shorter processes, and reductions in overall cell complexity\u003csup\u003e29,39,40\u003c/sup\u003e) were assessed at 1d following CFPI in the cortex and thalamus of animals treated with saline or Bup. Microglia within the thalamic domain demonstrated approximately 11 process endpoints/cell (saline=11.38\u0026plusmn;0.25; Bup=11.39\u0026plusmn;0.25), with somal perimeters of about 30\u0026micro;m\u003csup\u003e2 \u003c/sup\u003e(saline=29.20\u0026plusmn;0.49\u0026micro;m\u003csup\u003e2\u003c/sup\u003e; Bup=30.01\u0026plusmn;0.64\u0026micro;m\u003csup\u003e2\u003c/sup\u003e) and a maximum process segment length of around 20\u0026micro;m (saline=19.83\u0026plusmn;0.42\u0026micro;m; Bup=20.95\u0026plusmn;0.50\u0026micro;m) as well as an average cell complexity index of ~1.6 (saline1.606\u0026plusmn;0.01=; Bup=1.614\u0026plusmn;0.01 arbitrary units). There were no significant differences in any of these metrics between thalamic microglia in saline vs. Bup treated groups (Figure 5). Cortical microglia, however, demonstrated significant alterations in Bup treated animals as compared to saline. Cortical microglia in Bup treated animals had increased somal sizes (One-way-ANOVA, F\u003csub\u003e1,2627\u003c/sub\u003e=19.2, p=2.6x10\u003csup\u003e-12\u003c/sup\u003e; cortex saline vs. Bup p=2.3x10\u003csup\u003e-5\u003c/sup\u003e) and reduced process network complexity/microglia (One-way-ANOVA F\u003csub\u003e1,2627\u003c/sub\u003e=17.85, p=1.82x10\u003csup\u003e-11\u003c/sup\u003e; cortex saline vs. Bup p=0.019) compared to saline treated cortical microglia (Figure 5). All microglial morphological metrics demonstrated significant region-specific differences in which the cortex displayed reduced numbers of process endpoints (Two-way-ANOVA; Region F\u003csub\u003e1,2627\u003c/sub\u003e=64.89, p=1.19x10\u003csup\u003e-15\u003c/sup\u003e), larger soma sizes (Two-way-ANOVA; Region F\u003csub\u003e1,2627\u003c/sub\u003e=51.10, p=1.13x10\u003csup\u003e-12\u003c/sup\u003e), shorter maximum process length/cell (Two-way-ANOVA; Region F\u003csub\u003e1,2627\u003c/sub\u003e=19.67, p=1.0x10\u003csup\u003e-5\u003c/sup\u003e), and decreased process network complexity per microglia compared to the thalamus in either saline or Bup-treated animals (Two-way-ANOVA; Region F\u003csub\u003e1,2627\u003c/sub\u003e=44.05, p=3.87x10\u003csup\u003e-11\u003c/sup\u003e; Figure 5). There was a significant interaction between drug treatment and region for the microglial complexity index (Two-way-ANOVA Region*Treatment F\u003csub\u003e1,2627\u003c/sub\u003e=6.15, p=0.013).\u003c/p\u003e\n\u003cp\u003eAs opposed to microglia, cortical GFAP+ astrocytes, did not appear significantly altered 1d following TBI and Bup treatment as compared to saline treated animals (Figure 6). There were slight trends toward higher numbers of astrocytes/image (Two-way-ANOVA; Treatment F\u003csub\u003e1,16\u003c/sub\u003e=3.31, p=0.09) covering a greater percentage of the image (Two-way-ANOVA; Treatment F\u003csub\u003e1,16\u003c/sub\u003e=4.11, p=0.06) between saline and Bup treatment, however, neither of these trends were statistically significant. While astrocyte cell size was consistent between saline and Bup treatment groups in the cortex, thalamic astrocytes demonstrated significantly greater cell size in the Bup-treated animals compared to the saline treated animals at 1d post-injury (Two-way-ANOVA; Treatment F\u003csub\u003e1,16\u003c/sub\u003e=8.12, p=0.01; Figure 6G). There were also significant regional differences in the morphology of astrocytes in the control animals, with more astrocytes (Two-way-ANOVA; Region F=\u003csub\u003e1,16\u003c/sub\u003e=9.49, p=0.007) covering a larger proportion of the image (Two-way-ANOVA; Region F=\u003csub\u003e1,16\u003c/sub\u003e=8.43, p=0.01) in the thalamus of saline-treated control animals compared to the cortical astrocytes. These regional differences were not maintained following Bup treatment (Figure 6). The size of astrocytes, however, was (Two-way-ANOVA; Region F=\u003csub\u003e1,16\u003c/sub\u003e=24.04, p=1.59x10\u003csup\u003e-4\u003c/sup\u003e; Figure 6G). The circularity of astrocytes was also consistent between treatment groups and ROIs (Figure 6H). There were no interactions between region and treatment for any metrics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment with Buprenorphine altered myelin basic protein (MBP) isoforms but not overall MBP expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to assess the effect of Bup on myelin pathology at 1d post-CFPI, immunohistochemical labeling and Western blotting were performed to evaluate myelin integrity and the expression of myelin basic protein (MBP). There was no significant difference in the total number of intact myelin fibers (One-way-ANOVA; Region F=\u003csub\u003e1,16\u003c/sub\u003e=1.224, p=0.344) or myelin debris (One-way-ANOVA; Region F=\u003csub\u003e1,16\u003c/sub\u003e=0.558, p=0.653) between ROI or drug treatment groups (Figure 7E). Furthermore, overall MBP expression did not appear to be significantly altered with Bup treatment in either region (One-way-ANOVA; Region F=\u003csub\u003e1,20\u003c/sub\u003e=0.064, p=0.803). There did appear to be statistically significant regional differences, however, where overall expression of MBP was higher in the cortex than the thalamus, regardless of drug treatment group (One-way-ANOVA; Region F=\u003csub\u003e1,20\u003c/sub\u003e=33.576, p=0.000). As the different MBP isoforms are linked to different developmental stages of myelination\u003csup\u003e41\u003c/sup\u003e, the individual MBP isoforms (21.5 kDa, 18.5 kDa, 17.2 kDa, and 14.0 kDa) were also analyzed. Similar to analysis of the overall expression of MBP, the expression of individual MBP isoforms was not statistically different between Bup and saline treatment groups (Two-way ANOVA; Treatment 21.5 kDa: F=\u003csub\u003e1,20\u003c/sub\u003e=0.054, p=0.819, 18.5 kDa: F=\u003csub\u003e1,20\u003c/sub\u003e=0.127, p=0.727, 17.2 kDa: F=\u003csub\u003e1,20\u003c/sub\u003e=0.001, p=0.982, and 14.0 kDa: F=\u003csub\u003e1,20\u003c/sub\u003e=1.85, p=0.673; Figure 7G). There were, however, statistically significant regional differences for all isoforms (One-way ANOVA; 21.5 kDa: F=\u003csub\u003e1,20\u003c/sub\u003e=12.410, p=0.000, 18.5 kDa: F=\u003csub\u003e1,20\u003c/sub\u003e=6.363, p=0.005, 17.2 kDa: F=\u003csub\u003e1,20\u003c/sub\u003e=17.202, p=0.000, and 14.0 kDa: F=\u003csub\u003e1,20\u003c/sub\u003e=5.364, p=0.010) in which the expressions of all isoforms were higher in the cortex of either saline or Bup treatment group compared to the thalamus (Figure 7G).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBuprenorphine (Bup) is a semi-synthetic opioid which is a long-lasting and very effective partial agonist of the Mu opioid receptor (MOR) and an antagonist of the Kappa opioid receptor (KOR) and at higher doses the delta opioid receptor (DOR)\u003csup\u003e10,12\u003c/sup\u003e. The efficacy of low dose Bup in blocking the agonist effects of other opioids while also reducing withdrawal symptoms has made it a primary treatment for opioid use disorder \u003csup\u003e14\u003c/sup\u003e. Additionally, as there is a highly effective slow-release formula of Bup, that effectively alleviates pain for multiple days following a single subcutaneous injection, Bup is commonly used in experimental studies as a post-operative analgesic\u003csup\u003e16,19\u0026ndash;21\u003c/sup\u003e. To our knowledge there has only been one study evaluating the potential effects of Bup following CNS injury\u003csup\u003e42\u003c/sup\u003e. While this study found no differences in focal pathology following spinal cord injury, there have been no studies systematically assessing the potential effects of Bup-SR-Lab on the acute diffuse pathology produced by CNS injury. As the potential for analgesic-induced alterations on pathological progression give some investigators pause, the current study evaluated the effects of Bup-SR-Lab on acute physiological and diffuse pathological changes following TBI.\u003c/p\u003e\n\u003cp\u003eAlthough in previous studies Bup demonstrated physiological effects, such as respiratory depression and bradycardia\u003csup\u003e43\u003c/sup\u003e, we found that subcutaneous treatment with 1mg/kg Bup 15 min post-injury appeared to have no effect on systemic physiology. Specifically, heart rate and hemoglobin oxygen saturation were comparable between saline and Bup treated animals (Figure 1). While all animals in the current study were ventilated to maintain consistent respiratory rates, previous, studies found that Bup could act as a respiratory depressant. One study found that when rats were given a dose of Bup ranging from 1mg/kg to 3mg/kg respiratory rates decreased in a dose-dependent fashion\u003csup\u003e44\u003c/sup\u003e. Another study found that rats treated with 1.4mg/kg, 4.3mg/kg or 8.6mg/kg doses of Bup displayed a ceiling in Bup-induced respiratory depression after nearly 2 h of Bup infusion\u003csup\u003e45\u003c/sup\u003e. In another study, arterial pH and PaCO\u003csub\u003e2\u003c/sub\u003e were not altered in rats treated with 1.2mg/kg Bup-SR but did demonstrate lower arterial oxygen saturation compared to saline controls, indicating potential respiratory alterations\u003csup\u003e19\u003c/sup\u003e. This ceiling phenomenon, where respiratory depression reached its apparent maximum effect regardless of drug dose, is potentially attributed to Bup\u0026rsquo;s partial agonism at the MOR and may have prevented rapid changes in respiratory rate, as well as changes involving neural systems and behavioral processes\u003csup\u003e46\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhile all animals lost weight following TBI, animals treated with Bup demonstrated better maintenance of their body weight within the first day following CFPI compared to the saline control group (Figure 1). In a previous study using Bup-SR, weight was measured within the first 4 days after 0.3-4.5mg/kg doses were given to rats. Rats that were given 1.2mg/kg doses of Bup-SR maintained their weight but rats given 0.3mg/kg and 4.5mg/kg of Bup-SR gained or lost weight, respectively\u003csup\u003e16\u003c/sup\u003e. This indicates that there may be a dose-regulated response to Bup in regard to weight change in rats. Another study examining rabbits found that treatment of 0.03mg/kg of Bup every 12 h for 48 h 1d post-operatively experienced immediate weight loss post-operatively that sustained over the next four days, however, the rats displayed a slow return to baseline body weight by day 5 post-operatively\u003csup\u003e47\u003c/sup\u003e\u003cem\u003e.\u003c/em\u003e The reduced weight loss we observed at 1 day-post-injury may be attributed to the effects of Bup on gastrointestinal motility rather than suppressed appetite\u003csup\u003e48\u003c/sup\u003e. In a study examining the effects of 0.05mg/kg buprenorphine every 12 h for 48 h on rabbits found that buprenorphine induced gastrointestinal hypomotility of gut and delayed the passage of feces. Wheel running activity alters not only energy but food intake, neural systems involving stress response, and behavioral processes. A study found that voluntary wheel running activity was significantly lower at 24 h with Bup treatment but returned to baseline by 48 h\u003csup\u003e19\u003c/sup\u003e, which could impact body weight.\u003c/p\u003e\n\u003cp\u003eThe weight retention could also be mediated by the KOR. Kappa-opioid activation is known to cause stress, which is highly linked to weight loss\u003csup\u003e49\u003c/sup\u003e, but Bup acts as an antagonist of the KOR, which could reduce the effect of KOR-mediated weight loss. However, we were unable to find any studies investigating the association between KOR-activation and weight changes. Finally, while our study did not inspect the metabolism effects of Bup, it is known that Bup partially metabolizes to norbuprenorphine in the liver and both compounds are excreted as glucuronides. They then undergo enterohepatic circulation, where the drug and its metabolites can remain in intestinal circulation for days\u003csup\u003e50\u003c/sup\u003e. One study investigating Bup glucuronide metabolites discovered that two glucuronide metabolites of Bup are pharmacologically active\u003csup\u003e51\u003c/sup\u003e. This suggests that even as Bup is broken down in order to be eliminated from the body, its metabolites are still functioning and could have effects on weight loss at longer time points than the current study investigated.\u003c/p\u003e\n\u003cp\u003eAlthough both treatment groups presented DAI, there were no discernable differences in axonal injury between groups, indicated by the total number of APP+ axonal swellings in either the thalamus or cortex. There were also no significant indications of cell damge/death 1d post-CFPI in either region regardless of Bup treatment. , a previous study found that Bup administration prevented neuronal death and protected neurons in the medial and lateral regions of the thalamic reticular nucleus following resuscitation from cardiac arrest\u003csup\u003e52\u003c/sup\u003e. This region-specific finding was interesting as MORs are present in the medial and lateral regions of the thalamic reticular nucleus but not in the central regions\u003csup\u003e52\u003c/sup\u003e. This suggests that Bup may selectively protect regions in a MOR-mediated fashion. As the CFPI model used in our study does not precipitate cell death post-injury in either the cortex or the thalamus\u003csup\u003e27,53\u0026ndash;55\u003c/sup\u003e, it is unsurprising that Bup\u0026rsquo;s potential neuro-protective effects were not seen in the current study.\u003c/p\u003e\n\u003cp\u003eAcute neuroinflammation, however, does occur following CFPI as it does in human TBI\u003csup\u003e29,54,56\u0026ndash;60\u003c/sup\u003e. The primary cellular representatives for neuroinflammatory changes are microglia and astrocyte activations. The neuroinflammatory response of activated microglia and astrocytes occurs on a spectrum ranging from a pro-inflammatory phenotype, which is characterized by neurotoxic properties and release of neuroinflammatory cytokines, to an anti-inflammatory phenotype, which is characterized by the release of neurotrophic factors and anti-inflammatory cytokines that promote repair\u003csup\u003e61\u003c/sup\u003e. This correlates with the findings of higher cytokine expression (IL-1b, IL-4, IL-10, and IL-12) in the cortex, but our data found no discernable differences between treatment groups within either brain region analyzed. Corroboratively, studies have shown that animals treated with Bup, do not display systemic immunosuppression, which is common for MOR agonists, such as fentanyl\u003csup\u003e62\u003c/sup\u003e. One study, however, found higher levels of cytokines, such as IL-10 and TNFa, in the serum of animals treated with Bup compared to the control animals\u003csup\u003e63\u003c/sup\u003e, suggesting a potentially unique role of Bup on neuroinflammation as compared to other opioids. Another study recently found increased expression of IL-1b in the hippocampus of aged rats treated with morphine\u003csup\u003e64\u003c/sup\u003e weeks following surgical procedures, indicating that there might also be neuroinflammatory changes at later time points, which this study would not have captured. These possibilities will need to be further assessed at later time points following diffuse TBI, when neuroinflammatory responses are more robust\u003csup\u003e39,56\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eMicroglial and astrocyte activation can also be assessed via investigation of the morphological remodeling each cell type undergoes. Activated microglia demonstrate larger soma size, fewer process endpoints, shorter processes, and reductions in overall cell complexity as compared to non-activated ramified surveying microglia\u003csup\u003e29,40,54,65\u003c/sup\u003e. Activated astrocytes undergo hypertrophy and increased expression of the intermediate filament, GFAP\u003csup\u003e66,67\u003c/sup\u003e. Such activation-associated morphological changes of both microglia and astrocytes are seen in various brain regions following diffuse TBI\u003csup\u003e29,39,40,66,68,69\u003c/sup\u003e, however, the effects of Bup on such changes are not well understood. Both microglia and astrocytes express all three opioid receptors, suggesting that Bup could potentially play a role in modulation of microglial and/or astrocyte morphological changes associated with activation\u003csup\u003e10\u003c/sup\u003e. Our current studies found that Bup treatment was linked to a more activated morphological phenotype of microglia. Previous studies found that opioids given during development drastically reduce microglial process branching and hence the overall size and complexity of microglia; morphological changes indicative of microglial activation\u003csup\u003e9\u003c/sup\u003e. Other studies found that microglial activation is increased in a model of chronic morphine administration but can be reduced by the non-specific opioid receptor antagonist, Naloxone\u003csup\u003e70,71\u003c/sup\u003e. The effect of Bup on the immune system is not fully understood, but there is likely an interplay between its roles as a partial agonist at the MOR and antagonistic properties toward the other opioid receptors.\u003c/p\u003e\n\u003cp\u003eOur findings also indicate potential regional specificity of Bup-associated microglial activation. When comparing the microglial morphology between the two regions in both saline and Bup-treated animals, cortical microglia were found to exhibited larger soma, reduced process network complexity, shorter maximum process length/cell, and decreased numbers of process endpoints per microglia than the thalamus, regardless of treatment group. Additionally, microglia in the cortex demonstrated morphological changes associated with activation (larger cell bodies and reduced process network complexities), whereas these Bup-associated microglial changes were not observed in the thalamus.\u003c/p\u003e\n\u003cp\u003eAlternatively, Bup-treated rats demonstrated an increase in cell size, indicative of activation-linked hypertrophy, in the thalamus that was not discernable in the cortex of Bup-treated rats. Previous studies have found that astrocyte signaling is affected by buprenorphine. Treatment with Bup was associated with an upregulation of GFAP intensity\u003csup\u003e72\u003c/sup\u003e. Another study, however, found that Bup treatment reduced GFAP intensity in a model of morphine-induced dependence, demonstrating that astrocyte activation could be impacted by the interplay of multiple opioids\u003csup\u003e73\u003c/sup\u003e. Because Bup is a partial agonist of the MOR, at higher doses, Bup could induce an inhibition of the MOR that could impact microglial and astrocyte activation differentially\u003csup\u003e74\u003c/sup\u003e. The MOR is more highly expressed in the thalamus as compared to the lateral neocortex, which has low to moderate opioid receptor expression\u003csup\u003e75\u003c/sup\u003e. Therefore, the regional-specific differences between Bup effects on microglial and astrocyte morphologies could be linked to differences in the opioid receptor profiles between these two brain regions.\u003c/p\u003e\n\u003cp\u003eA previous study found that Bup exposure during development had a significant impact on myelin protein expression. Specifically, it was found that a low dose of 0.3mg/kg Bup given to early postnatal rat pups resulted in significantly increased expression of all MBP isoforms\u003csup\u003e23\u003c/sup\u003e. However, when given at a higher dose of 1mg/kg, expression of MBP was delayed\u003csup\u003e74\u003c/sup\u003e. While our study utilized a dose of 1mg/kg Bup-SR-Lab, we found no change in overall MBP expression between the Bup treatment group and the saline treatment group. We did, however, observe regional differences between the cortex and, with higher expression of MBP in the cortex than in the thalamus (Figure 7F). This could be due to the adult age at which our study was conducted, in which developmental myelination has already occurred and delays in MBP expression upon injury and subsequent repair might be more subtle.\u003c/p\u003e\n\u003cp\u003eThe myelin protein, MBP, consists of four major isoforms with molecular weights of 21.5 kDa, 18.5 kDa, 17.2 kDa, and 14.0 kDa, produced via alternate splicing of the primary MBP transcript\u003csup\u003e41\u003c/sup\u003e. One study investigating the degradation of MBP following TBI reported that all four major isoforms of MBP were degraded within hours following injury and intact protein levels did not return to base levels for 3-5 days post-injury\u003csup\u003e76\u003c/sup\u003e. This study observed significant proteolysis of MBP in the cortex hours post-injury but found MBP breakdown in the hippocampus was more delayed with MBP breakdown not peaking until 48 h after TBI potentially due to differences in the compression-induced contusion force\u003csup\u003e76\u003c/sup\u003e. It is possible that in the current study, we observed higher levels of MBP expression in the cortex as compared to the thalamus due to such differences in biomechanical forces, however, in this study we were assessing MBP and not the breakdown of MBP. It is also possible that the observed difference in MBP expression in the cortex vs. the thalamus is reflective of the proportion of myelinated fibers within each region. Ultimately, it is likely that myelin pathology alterations could evolve over longer post-injury time-points following CFPI.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe findings of this initial study show that preclinical use of Bup-SR-Lab has little effect on acute pathology following diffuse brain injury. However, because only one acute time point was observed in this study, future studies are required to examine potential long-term differences in physiological and pathological outcomes following TBI. Additionally, as morphological aspects of both microglia and astrocytes were found to be changed by Bup-SR-Lab treatment, metanalytical studies assessing these parameters should be aware of the potential effects of post-injury Bup treatment on these outcome metrics. These subtle changes, however, would not preclude the use of Bup-SR-Lab for post-TBI pain management in primary acute-survival animal studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTBI: Traumatic brain injury\u003c/p\u003e\n\u003cp\u003eBup-SR-Lab: buprenorphine-Slow-release-Lab\u003c/p\u003e\n\u003cp\u003eCFPI: central fluid percussion injury\u003c/p\u003e\n\u003cp\u003eROI: region of interest\u003c/p\u003e\n\u003cp\u003eMBP: myelin basic protein\u003c/p\u003e\n\u003cp\u003eDAI: diffuse axonal injury\u003c/p\u003e\n\u003cp\u003eFITBIR: Federal Interagency Traumatic Brain Injury Research\u003c/p\u003e\n\u003cp\u003eBup: buprenorphine\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E: hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eCat: catalogue\u003c/p\u003e\n\u003cp\u003eSEM: standard error of the mean\u003c/p\u003e\n\u003cp\u003eAPP: amyloid precursor protein\u003c/p\u003e\n\u003cp\u003eMOR: Mu opioid receptor\u003c/p\u003e\n\u003cp\u003eKOR: Kappa opioid receptor\u003c/p\u003e\n\u003cp\u003eDOR: delta opioid receptor\u003c/p\u003e\n\u003cp\u003eCNS: central nervous system\u003c/p\u003e\n\u003cp\u003eGFAP: glial fibrillary acidic protein\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiments were conducted in accordance with the Virginia Commonwealth University institutional ethical guidelines concerning the care and use of laboratory animals (Institutional Animal Care and Use Committee, Virginia Commonwealth University), which adhere to regulations including, but not limited to, those set forth in the \u0026ldquo;Guide for the Care and Use of Laboratory Animals: 8th Edition\u0026rdquo; (National Research Council).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grant NINDS R01NS096143.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJR conducted the myelin pathology and MBP expression analyses and wrote the manuscript. PS carried out the histological assessments and wrote the paper. SL, BP and KG carried out the histological assessments. ADL developed the histological assessments, conceived, designed and coordinated the study and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to recognize Judy Williamson, Frances White and Susan Walker for invaluable technical assistance. This work was supported by NINDS grant 1R01NS096143.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCoronado, V. G. \u003cem\u003eet al.\u003c/em\u003e Surveillance for traumatic brain injury-related deaths--United States, 1997-2007. \u003cem\u003eMMWR. Surveill. Summ.\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 1\u0026ndash;32 (2011).\u003c/li\u003e\n\u003cli\u003eOrtiz-Prado, E. \u003cem\u003eet al.\u003c/em\u003e A Nationwide Study of Incidence and Mortality Due to Traumatic Brain Injury in Ecuador (2004-2016). \u003cem\u003eNeuroepidemiology\u003c/em\u003e 1\u0026ndash;12 (2019) doi:10.1159/000502580.\u003c/li\u003e\n\u003cli\u003eBramlett, H. M. \u0026amp; Dietrich, W. D. Pathophysiology of cerebral ischemia and brain trauma: similarities and differences. \u003cem\u003eJ Cereb Blood Flow Metab\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 133\u0026ndash;150 (2004).\u003c/li\u003e\n\u003cli\u003eHome | FITBIR. (2019).\u003c/li\u003e\n\u003cli\u003eNational Research Council. \u003cem\u003eGUIDE LABORATORY ANIMALS FOR THE CARE AND USE OF Eighth Edition Committee for the Update of the Guide for the Care and Use of Laboratory Animals Institute for Laboratory Animal Research Division on Earth and Life Studies\u003c/em\u003e. (2011).\u003c/li\u003e\n\u003cli\u003eNational Institutes of Health. Public Health Service Policy on Humane Care and Use of Laboratory Animals. \u003cem\u003eOffice Lab. Anim. Welf.\u003c/em\u003e 2002 (2013).\u003c/li\u003e\n\u003cli\u003eSun, J., Guo, W. \u0026amp; Du, X. Buprenorphine differentially affects M1- and M2-polarized macrophages from human umbilical cord blood. \u003cem\u003eEur. Cytokine Netw.\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 85\u0026ndash;92 (2017).\u003c/li\u003e\n\u003cli\u003eCorkrum, M., Rothwell, P. E., Thomas, M. J., Kofuji, P. \u0026amp; Araque, A. Opioid-Mediated Astrocyte\u0026ndash;Neuron Signaling in the Nucleus Accumbens. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 586 (2019).\u003c/li\u003e\n\u003cli\u003eJantzie, L. L. \u003cem\u003eet al.\u003c/em\u003e Prenatal opioid exposure: The next neonatal neuroinflammatory disease. \u003cem\u003eBrain. Behav. Immun.\u003c/em\u003e \u003cstrong\u003e84\u003c/strong\u003e, 45\u0026ndash;58 (2020).\u003c/li\u003e\n\u003cli\u003eMurphy, A. \u003cem\u003eet al.\u003c/em\u003e The Effects of Opioids on HIV Neuropathogenesis. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eIlb\u0026auml;ck, N.-G., Siller, M. \u0026amp; St\u0026aring;lhandske, T. Effects of buprenorphine on body temperature, locomotor activity and cardiovascular function when assessed by telemetric monitoring in rats. \u003cem\u003eLab. Anim.\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 149\u0026ndash;160 (2008).\u003c/li\u003e\n\u003cli\u003eVilliger, J. W. \u0026amp; Taylor, K. M. Buprenorphine: Characteristics of binding sites in the rat central nervous system. \u003cem\u003eLife Sci.\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 2699\u0026ndash;2708 (1981).\u003c/li\u003e\n\u003cli\u003eBoas, R. A. \u0026amp; Villiger, J. W. \u003cem\u003eCLINICAL ACTIONS OF FENTANYL AND BUPRENORPHINE The Significance of Receptor Binding\u003c/em\u003e. \u003cem\u003eBr.J. Anaesth\u003c/em\u003e vol. 57 (1985).\u003c/li\u003e\n\u003cli\u003eGreenwald, M. K., Comer, S. D. \u0026amp; Fiellin, D. A. Buprenorphine maintenance and mu-opioid receptor availability in the treatment of opioid use disorder: Implications for clinical use and policy. \u003cem\u003eDrug Alcohol Depend.\u003c/em\u003e \u003cstrong\u003e144\u003c/strong\u003e, 1\u0026ndash;11 (2014).\u003c/li\u003e\n\u003cli\u003eWelsh, C. \u0026amp; Valadez-Meltzer, A. Buprenorphine: a (relatively) new treatment for opioid dependence. \u003cem\u003ePsychiatry (Edgmont).\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 29\u0026ndash;39 (2005).\u003c/li\u003e\n\u003cli\u003eChum, H. H. \u003cem\u003eet al.\u003c/em\u003e Antinociceptive effects of sustained-release buprenorphine in a model of incisional pain in rats (Rattus norvegicus). \u003cem\u003eJ. Am. Assoc. Lab. Anim. Sci.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 193\u0026ndash;197 (2014).\u003c/li\u003e\n\u003cli\u003eFalcon, E. \u003cem\u003eet al.\u003c/em\u003e Antidepressant-like Effects of Buprenorphine are Mediated by Kappa Opioid Receptors. \u003cem\u003eNeuropsychopharmacology\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 2344\u0026ndash;2351 (2016).\u003c/li\u003e\n\u003cli\u003eKendall, L. V \u003cem\u003eet al.\u003c/em\u003e Pharmacokinetics of sustained-release analgesics in mice. \u003cem\u003eJ. Am. Assoc. Lab. Anim. Sci.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 478\u0026ndash;484 (2014).\u003c/li\u003e\n\u003cli\u003eJohnson, R. A. Voluntary running-wheel activity, arterial blood gases, and thermal antinociception in rats after 3 buprenorphine formulations. \u003cem\u003eJ. Am. Assoc. Lab. Anim. Sci.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 306\u0026ndash;311 (2016).\u003c/li\u003e\n\u003cli\u003eSeymour, T. L. \u003cem\u003eet al.\u003c/em\u003e Postoperative analgesia due to sustained-release buprenorphine, sustained-release meloxicam, and carprofen gel in a model of incision pain in rats (Rattus norvegicus). \u003cem\u003eJaalas\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 300\u0026ndash;305 (2016).\u003c/li\u003e\n\u003cli\u003eFoley, P. L., Liang, H. \u0026amp; Crichlow, A. R. Evaluation of a sustained-release formulation of buprenorphine for analgesia in rats. \u003cem\u003eJ. Am. Assoc. Lab. Anim. Sci.\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 198\u0026ndash;204 (2011).\u003c/li\u003e\n\u003cli\u003eDeClue, A. E. \u003cem\u003eet al.\u003c/em\u003e Effects of opioids on phagocytic function, oxidative burst capacity, cytokine production and apoptosis in canine leukocytes. \u003cem\u003eVet. J.\u003c/em\u003e \u003cstrong\u003e200\u003c/strong\u003e, 270\u0026ndash;275 (2014).\u003c/li\u003e\n\u003cli\u003eSanchez, E. S., Bigbee, J. W., Fobbs, W., Robinson, S. E. \u0026amp; Sato-Bigbee, C. Opioid addiction and pregnancy: Perinatal exposure to buprenorphine affects myelination in the developing brain. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 1017\u0026ndash;1027 (2008).\u003c/li\u003e\n\u003cli\u003eLafrenaye, A. D., Krahe, T. E. \u0026amp; Povlishock, J. T. Moderately Elevated Intracranial Pressure after Diffuse Traumatic Brain Injury is Associated with Exacerbated Neuronal Pathology and Behavioral Morbidity in the Rat. \u003cem\u003eJ. Cereb. Blood Flow Metab.\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 1628\u0026ndash;1636 (2014).\u003c/li\u003e\n\u003cli\u003eHernandez, M. L., Chatlos, T., Gorse, K. M. \u0026amp; Lafrenaye, A. D. Neuronal Membrane Disruption Occurs Late Following Diffuse Brain Trauma in Rats and Involves a Subpopulation of NeuN Negative Cortical Neurons. \u003cem\u003eFront. Neurol.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003cli\u003eDixon, C. E. \u003cem\u003eet al.\u003c/em\u003e A fluid percussion model of experimental brain injury in the rat. \u003cem\u003eJ. Neurosurg.\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 110\u0026ndash;119 (1987).\u003c/li\u003e\n\u003cli\u003eGorse, K. M. \u0026amp; Lafrenaye, A. D. The importance of inter-species variation in traumatic brain injury-induced alterations of microglial-axonal interactions. \u003cem\u003eFront. Neurol.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 778 (2018).\u003c/li\u003e\n\u003cli\u003eLafrenaye, A. D., McGinn, M. J. \u0026amp; Povlishock, J. T. Increased intracranial pressure after diffuse traumatic brain injury exacerbates neuronal somatic membrane poration but not axonal injury: Evidence for primary intracranial pressure-induced neuronal perturbation. \u003cem\u003eJ. Cereb. Blood Flow Metab.\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 1919\u0026ndash;1932 (2012).\u003c/li\u003e\n\u003cli\u003eLafrenaye, A. D., Todani, M., Walker, S. A. \u0026amp; Povlishock, J. T. Microglia processes associate with diffusely injured axons following mild traumatic brain injury in the micro pig. \u003cem\u003eJ. Neuroinflammation\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 186 (2015).\u003c/li\u003e\n\u003cli\u003eSimon, C. M., Sharif, S., Tan, R. P. \u0026amp; LaPlaca, M. C. Spinal cord contusion causes acute plasma membrane damage. \u003cem\u003eJ. Neurotrauma\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 563\u0026ndash;574 (2009).\u003c/li\u003e\n\u003cli\u003eJohnson, V. E., Stewart, W. \u0026amp; Smith, D. H. Axonal pathology in traumatic brain injury. \u003cem\u003eExp. Neurol.\u003c/em\u003e \u003cstrong\u003e246\u003c/strong\u003e, 35\u0026ndash;43 (2013).\u003c/li\u003e\n\u003cli\u003eBenson, C. Diffuse Axonal Injury. in \u003cem\u003eEncyclopedia of the Neurological Sciences\u003c/em\u003e 998\u0026ndash;999 (Elsevier, 2014). doi:10.1016/B978-0-12-385157-4.00326-2.\u003c/li\u003e\n\u003cli\u003eSmith, D. H., Meaney, D. F. \u0026amp; Shull, W. H. Diffuse axonal injury in head trauma. \u003cem\u003eJ Head Trauma Rehabil\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 307\u0026ndash;316 (2003).\u003c/li\u003e\n\u003cli\u003eGeddes, D. M., LaPlaca, M. C. \u0026amp; Cargill, R. S. Susceptibility of hippocampal neurons to mechanically induced injury. \u003cem\u003eExp. Neurol.\u003c/em\u003e \u003cstrong\u003e184\u003c/strong\u003e, 420\u0026ndash;427 (2003).\u003c/li\u003e\n\u003cli\u003eLaPlaca, M. C. \u003cem\u003eet al.\u003c/em\u003e Mechanoporation is a potential indicator of tissue strain and subsequent degeneration following experimental traumatic brain injury. \u003cem\u003eClin. Biomech.\u003c/em\u003e \u003cstrong\u003e64\u003c/strong\u003e, 2\u0026ndash;13 (2019).\u003c/li\u003e\n\u003cli\u003eWhalen, M. J. \u003cem\u003eet al.\u003c/em\u003e Acute plasmalemma permeability and protracted clearance of injured cells after controlled cortical impact in mice. \u003cem\u003eJ. Cereb. Blood Flow Metab.\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 490\u0026ndash;505 (2008).\u003c/li\u003e\n\u003cli\u003eFarkas, O., Lifshitz, J. \u0026amp; Povlishock, J. T. Mechanoporation Induced by Diffuse Traumatic Brain Injury: An Irreversible or Reversible Response to Injury? \u003cem\u003eJ. Neurosci.\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 3130 LP \u0026ndash; 3140 (2006).\u003c/li\u003e\n\u003cli\u003ePrado, G. R. \u0026amp; LaPlaca, M. C. Neuronal Plasma Membrane Integrity is Transiently Disturbed by Traumatic Loading. \u003cem\u003eNeurosci. Insights\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eLoane, D. J., Kumar, A., Stoica, B. A., Cabatbat, R. \u0026amp; Faden, A. I. Progressive Neurodegeneration After Experimental Brain Trauma. \u003cem\u003eJ. Neuropathol. Exp. Neurol.\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 14\u0026ndash;29 (2014).\u003c/li\u003e\n\u003cli\u003eByrnes, K. R., Loane, D. J., Stoica, B. a, Zhang, J. \u0026amp; Faden, A. I. Delayed mGluR5 activation limits neuroinflammation and neurodegeneration after traumatic brain injury. \u003cem\u003eJ. Neuroinflammation\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 43 (2012).\u003c/li\u003e\n\u003cli\u003eAkiyama, K., Ichinose, S., Omori, A., Sakurai, Y. \u0026amp; Asou, H. Study of expression of myelin basic proteins (MBPs) in developing rat brain using a novel antibody reacting with four major isoforms of MBP. \u003cem\u003eJ. Neurosci. Res.\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 19\u0026ndash;28 (2002).\u003c/li\u003e\n\u003cli\u003eSantiago, J. M. \u003cem\u003eet al.\u003c/em\u003e Molecular, anatomical, physiological, and behavioral studies of rats treated with buprenorphine after spinal cord injury. \u003cem\u003eJ. Neurotrauma\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 1783\u0026ndash;1793 (2009).\u003c/li\u003e\n\u003cli\u003eDooley, S. B. \u003cem\u003eet al.\u003c/em\u003e Pharmacokinetics and pharmacodynamics of buprenorphine and sustained-release buprenorphine after administration to adult alpacas. \u003cem\u003eAm. J. Vet. Res.\u003c/em\u003e \u003cstrong\u003e78\u003c/strong\u003e, 321\u0026ndash;329 (2017).\u003c/li\u003e\n\u003cli\u003eGuarnieri, M. \u003cem\u003eet al.\u003c/em\u003e Safety and efficacy of buprenorphine for analgesia in laboratory mice and rats. \u003cem\u003eLab Animal\u003c/em\u003e vol. 41 337\u0026ndash;343 (2012).\u003c/li\u003e\n\u003cli\u003eDahan, A. \u003cem\u003eet al.\u003c/em\u003e Comparison of the respiratory effects of intravenous buprenorphine and fentanyl in humans and rats. \u003cem\u003eBr. J. Anaesth.\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 825\u0026ndash;834 (2005).\u003c/li\u003e\n\u003cli\u003eDahan, A. \u003cem\u003eet al.\u003c/em\u003e Buprenorphine induces ceiling in respiratory depression but not in analgesia. \u003cem\u003eBr. J. Anaesth.\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 627\u0026ndash;632 (2006).\u003c/li\u003e\n\u003cli\u003eCooper, C. S., Metcalf-Pate, K. A., Barat, C. E., Cook, J. A. \u0026amp; Scorpio, D. G. Comparison of side effects between buprenorphine and meloxicam used postoperatively in Dutch belted rabbits (Oryctolagus cuniculus). \u003cem\u003eJ. Am. Assoc. Lab. Anim. Sci.\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 279\u0026ndash;285 (2009).\u003c/li\u003e\n\u003cli\u003eMartin-Flores, M. \u003cem\u003eet al.\u003c/em\u003e Effects of buprenorphine, methylnaltrexone, and their combination on gastrointestinal transit in healthy New Zealand white rabbits. \u003cem\u003eJ. Am. Assoc. Lab. Anim. Sci.\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 155\u0026ndash;159 (2017).\u003c/li\u003e\n\u003cli\u003eCarroll, F. I. \u0026amp; Carlezon, W. A. Development of \u0026kappa; opioid receptor antagonists. \u003cem\u003eJ. Med. Chem.\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 2178\u0026ndash;2195 (2013).\u003c/li\u003e\n\u003cli\u003eElkader, A. \u0026amp; Sproule, B. \u003cem\u003eBuprenorphine Clinical Pharmacokinetics in the Treatment of Opioid Dependence\u003c/em\u003e. \u003cem\u003eClin Pharmacokinet\u003c/em\u003e vol. 44 https://link-springer-com.proxy.library.vcu.edu/content/pdf/10.2165%2F00003088-200544070-00001.pdf (2005).\u003c/li\u003e\n\u003cli\u003eBrown, S. M., Holtzman, M., Kim, T. \u0026amp; Kharasch, E. D. \u003cem\u003eBuprenorphine Metabolites, Buprenorphine-3-glucuronide and Norbuprenorphine-3-glucuronide, Are Biologically Active\u003c/em\u003e. http://pubs.asahq.org/anesthesiology/article-pdf/115/6/1251/256341/0000542-201112000-00020.pdf (2011).\u003c/li\u003e\n\u003cli\u003eSabol Jones, M. K. \u0026amp; Ross, D. T. The partial \u0026mu; opiate agonist buprenorphine protects a sub-population of thalamic reticular neurons following cardiac arrest in rats. \u003cem\u003eNeurosci. Lett.\u003c/em\u003e \u003cstrong\u003e185\u003c/strong\u003e, 91\u0026ndash;94 (1995).\u003c/li\u003e\n\u003cli\u003eLafrenaye, A. D., Krahe, T. E. \u0026amp; Povlishock, J. T. Moderately elevated intracranial pressure after diffuse traumatic brain injury is associated with exacerbated neuronal pathology and behavioral morbidity in the rat. \u003cem\u003eJ. Cereb. Blood Flow Metab.\u003c/em\u003e 1\u0026ndash;9 (2014) doi:10.1038/jcbfm.2014.122.\u003c/li\u003e\n\u003cli\u003eThomas, T. C. \u003cem\u003eet al.\u003c/em\u003e Does time heal all wounds? Experimental diffuse traumatic brain injury results in persisting histopathology in the thalamus. \u003cem\u003eBehav. Brain Res.\u003c/em\u003e \u003cstrong\u003e340\u003c/strong\u003e, 137\u0026ndash;146 (2018).\u003c/li\u003e\n\u003cli\u003eLafrenaye, A. D., McGinn, M. J. \u0026amp; Povlishock, J. T. Increased intracranial pressure after diffuse traumatic brain injury exacerbates neuronal somatic membrane poration but not axonal injury: evidence for primary intracranial pressure-induced neuronal perturbation. \u003cem\u003eJ Cereb Blood Flow Metab\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 1919\u0026ndash;1932 (2012).\u003c/li\u003e\n\u003cli\u003eKelley, B. J., Lifshitz, J. \u0026amp; Povlishock, J. T. Neuroinflammatory responses after experimental diffuse traumatic brain injury. \u003cem\u003eJ. Neuropathol. Exp. Neurol.\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 989\u0026ndash;1001 (2007).\u003c/li\u003e\n\u003cli\u003eWitcher, K. G. \u003cem\u003eet al.\u003c/em\u003e Traumatic brain injury-induced neuronal damage in the somatosensory cortex causes formation of rod-shaped microglia that promote astrogliosis and persistent neuroinflammation. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 2719\u0026ndash;2736 (2018).\u003c/li\u003e\n\u003cli\u003eRowe, R. K. \u003cem\u003eet al.\u003c/em\u003e Diffuse traumatic brain injury induces prolonged immune dysregulation and potentiates hyperalgesia following a peripheral immune challenge. \u003cem\u003eMol. Pain\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1\u0026ndash;12 (2016).\u003c/li\u003e\n\u003cli\u003eCoughlin, J. M. \u003cem\u003eet al.\u003c/em\u003e Neuroinflammation and brain atrophy in former NFL players: An in vivo multimodal imaging pilot study. \u003cem\u003eNeurobiol. Dis.\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 58\u0026ndash;65 (2015).\u003c/li\u003e\n\u003cli\u003eVel\u0026aacute;zquez, A., Ortega, M., Rojas, S., Gonz\u0026aacute;lez-Oliv\u0026aacute;n, F. J. \u0026amp; Rodr\u0026iacute;guez-Baeza, A. Widespread microglial activation in patients deceased from traumatic brain injury. \u003cem\u003eBrain Inj.\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 1126\u0026ndash;1133 (2015).\u003c/li\u003e\n\u003cli\u003eZiebell, J. M. \u0026amp; Morganti-Kossmann, M. C. Involvement of Pro- and Anti-Inflammatory Cytokines and Chemokines in the Pathophysiology of Traumatic Brain Injury. \u003cem\u003eNeurotherapeutics\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 22\u0026ndash;30 (2010).\u003c/li\u003e\n\u003cli\u003eRoy, S. \u003cem\u003eet al.\u003c/em\u003e Do All Opioid Drugs Share the Same Immunomodulatory Properties? A Review From Animal and Human Studies. \u003cem\u003eFront. Immunol. | www.frontiersin.org\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 2914 (2019).\u003c/li\u003e\n\u003cli\u003eF\u0026eacute;lix, N. M. \u003cem\u003eet al.\u003c/em\u003e Effects of buprenorphine in the adrenal, thyroid, and cytokine intra-operative responses in a rat model (Rattus norvegicus): a preliminary study. doi:10.22038/IJBMS.2017.8576.\u003c/li\u003e\n\u003cli\u003eMuscat, S. M. \u003cem\u003eet al.\u003c/em\u003e Post-operative cognitive dysfunction is made persistent with morphine treatment in aged rats. \u003cem\u003eNeurobiol. Aging\u003c/em\u003e (2020) doi:10.1016/j.neurobiolaging.2020.11.008.\u003c/li\u003e\n\u003cli\u003eMorrison, H. W. \u0026amp; Filosa, J. a. A quantitative spatiotemporal analysis of microglia morphology during ischemic stroke and reperfusion. \u003cem\u003eJ. Neuroinflammation\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 4 (2013).\u003c/li\u003e\n\u003cli\u003eCl\u0026eacute;ment, T. \u003cem\u003eet al.\u003c/em\u003e Juvenile mild traumatic brain injury elicits distinct spatiotemporal astrocyte responses. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 528\u0026ndash;542 (2020).\u003c/li\u003e\n\u003cli\u003ePekny, M. \u0026amp; Nilsson, M. Astrocyte activation and reactive gliosis. \u003cem\u003eGlia\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 427\u0026ndash;434 (2005).\u003c/li\u003e\n\u003cli\u003eHall, K. D. \u0026amp; Lifshitz, J. Diffuse traumatic brain injury initially attenuates and later expands activation of the rat somatosensory whisker circuit concomitant with neuroplastic responses. \u003cem\u003eBrain Res.\u003c/em\u003e \u003cstrong\u003e1323\u003c/strong\u003e, 161\u0026ndash;73 (2010).\u003c/li\u003e\n\u003cli\u003eLafrenaye, A. D. \u003cem\u003eet al.\u003c/em\u003e Circulating GFAP and Iba-1 levels are associated with pathophysiological sequelae in the thalamus in a pig model of mild TBI. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 13369 (2020).\u003c/li\u003e\n\u003cli\u003eLi, Z., Jia, X., Peng, X. \u0026amp; Gao, F. The interaction between spinal pdgfr\u0026beta; and \u0026mu; opioid receptor in the activation of microglia in morphine-tolerant rats. \u003cem\u003eJ. Pain Res.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1803\u0026ndash;1810 (2020).\u003c/li\u003e\n\u003cli\u003eCarranza-Aguilar, C. J. \u003cem\u003eet al.\u003c/em\u003e Morphine and Fentanyl Repeated Administration Induces Different Levels of NLRP3-Dependent Pyroptosis in the Dorsal Raphe Nucleus of Male Rats via Cell-Specific Activation of TLR4 and Opioid Receptors NLRP3 \u0026middot; Opioids \u0026middot; Glia \u0026middot; TLR4 \u0026middot; NF-\u0026kappa;B \u0026middot; Serotonin. \u003cem\u003eCell. Mol. Neurobiol.\u003c/em\u003e doi:10.1007/s10571-020-00957-5.\u003c/li\u003e\n\u003cli\u003eGerhold, K. J., Drdla-Schutting, R., Honsek, S. D., Forsthuber, L. \u0026amp; Sandk\u0026uuml;hler, J. Pronociceptive and antinociceptive effects of buprenorphine in the spinal cord dorsal horn cover a dose range of four orders of magnitude. \u003cem\u003eJ. Neurosci.\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 9580\u0026ndash;9594 (2015).\u003c/li\u003e\n\u003cli\u003eWu, F. X. \u003cem\u003eet al.\u003c/em\u003e Dezocine Alleviates Morphine-Induced Dependence in Rats. \u003cem\u003eAnesth. Analg.\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, 1328\u0026ndash;1335 (2019).\u003c/li\u003e\n\u003cli\u003eVestal-Laborde, A. A., Eschenroeder, A. C., Bigbee, J. W., Robinson, S. E. \u0026amp; Sato-Bigbee, C. The opioid system and brain development: Effects of methadone on the oligodendrocyte lineage and the early stages of myelination. \u003cem\u003eDev. Neurosci.\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 409\u0026ndash;421 (2014).\u003c/li\u003e\n\u003cli\u003eLe Merrer, J., Becker, J. A. J., Befort, K. \u0026amp; Kieffer, B. L. Reward processing by the opioid system in the brain. \u003cem\u003ePhysiological Reviews\u003c/em\u003e vol. 89 1379\u0026ndash;1412 (2009).\u003c/li\u003e\n\u003cli\u003eMing, C. L. \u003cem\u003eet al.\u003c/em\u003e Extensive degradation of myelin basic protein isoforms by calpain following traumatic brain injury. \u003cem\u003eJ. Neurochem.\u003c/em\u003e \u003cstrong\u003e98\u003c/strong\u003e, 700\u0026ndash;712 (2006).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Traumatic brain injury, buprenorphine, Bup-SR-Lab, microglia, astrocyte, membrane disruption, myelin ","lastPublishedDoi":"10.21203/rs.3.rs-138979/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-138979/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Traumatic brain injury (TBI) is a common phenomenon, accounting for significant cost and adverse health effects. While there is information about focal pathologies following TBI, knowledge of more diffuse processes is lacking, particularly regarding how analgesics affect this pathology. As buprenorphine is the most commonly used analgesic in experimental TBI models, this study investigated the acute effects of the opioid analgesic buprenorphine (Bup-SR-Lab) on diffuse neuronal/glial pathology, neuroinflammation, cell damage, and systemic physiology. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We utilized a model of central fluid percussion injury (CFPI) in adult male rats treated with a single subcutaneous bolus of Bup-SR-Lab or saline 15min post-injury. Microscopic assessments were performed at 1 day post-injury. Cell impermeable dextran was infused intraventricularly prior to sacrifice to assess neuronal membrane disruption. Axonal injury was assessed by investigating labeling of the anterogradely transported amyloid precursor protein. Neuroinflammation was assessed by analyzing Iba-1+ microglial and GFAP+ astrocyte histological/morphological features as well as cytokine levels in both regions of interest (ROIs). Myelin pathology was assessed by evaluating the expression of myelin basic protein (MBP) and the propensity of MBP+ myelin debris. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Acute physiologic data showed no difference between groups except for reduction in weight loss following cFPI in Bup treated animals compared to saline. There were no discernable differences in axonal injury or membrane disruption between treatment groups. Cytokine levels were consistent between Bup and saline treated animals, however, microglia and astrocytes revealed region specific histological changes at 1d following Bup treatment. Myelin integrity and overall MBP expression showed no differences between Bup and saline treated animals, but there were significant regional differences in MBP expression between the cortex and thalamus.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: These data suggest effects of Bup treatment on weight following CFPI and potential regional specificity of Bup-associated microglial and astrocyte alterations, but very little change in other acute pathology at 1-day post-injury. Overall, this preliminary study indicates that use of Bup-SR-Lab in preclinical work does have effects on acute glial pathology, however, longer term studies will be needed to assess potential effects of Bup treatment on more chronic pathological progressions.\u003c/p\u003e","manuscriptTitle":"Buprenorphine Alters Microglia and Astrocytes Acutely Following Diffuse Traumatic Brain Injury.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-07 22:52:00","doi":"10.21203/rs.3.rs-138979/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-02-03T09:00:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-01-22T16:23:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81683af3-337e-48ae-a269-7144587628bc","date":"2021-01-11T18:06:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-01-06T16:37:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-01-06T16:33:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-01-06T05:30:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-01-04T08:06:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2020-12-31T16:37:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"03d7b2c0-9d05-4fae-8278-b4e9af7b8f05","owner":[],"postedDate":"January 7th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1759196,"name":"Cellular \u0026 Molecular Neuroscience"}],"tags":[],"updatedAt":"2021-08-18T19:13:48+00:00","versionOfRecord":{"articleIdentity":"rs-138979","link":"https://doi.org/10.1038/s41598-021-88030-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2021-04-21 19:02:22","publishedOnDateReadable":"April 21st, 2021"},"versionCreatedAt":"2021-01-07 22:52:00","video":"","vorDoi":"10.1038/s41598-021-88030-z","vorDoiUrl":"https://doi.org/10.1038/s41598-021-88030-z","workflowStages":[]},"version":"v1","identity":"rs-138979","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-138979","identity":"rs-138979","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}