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% O2/70% room air. Animals were then intubated and ventilated with 1.5%-2.5% isoflurane in 30% O2 and 70% room air throughout the duration of the surgery, injury, and physiologic monitoring. Body temperature was maintained at 37oC 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 placement24,25. 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 previously24–27. 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% O2/70% room air followed by maintenance dose of 2% isoflurane in 30% O2/70% 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 previously25, 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 μm from bregma to ∼4.0 mm posterior to bregma. Sections were collected serially in 12-well plates and stored in MIllonig’s buffer at 4oC. 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 previously24,25,28. 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 described24,29. 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% H202/0.3% 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 dextran24,25,30, 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 µm2. To assess myelin fibers the analysis settings were as follows, circularity= 0.0-0.1 and particle size= 25-infinity µm2. 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.