Platelet-Like Particles Reduce Coagulopathy-Related and Neuroinflammatory Pathologies Post-Experimental Traumatic Brain Injury

Abstract

Coagulopathy may occur following traumatic brain injury (TBI) thereby negatively affecting patient outcomes. Here, we investigate the use of platelet-like particles (PLPs), poly(N-isopropylacrylamide-co-acrylic-acid) microgels conjugated with a fibrin-specific antibody, to improve hemostasis post-TBI. The objective of this study was to diminish coagulopathy in a mouse TBI model (controlled cortical impact) via PLP treatment, and subsequently decrease blood-brain barrier (BBB) permeability and neuroinflammation. Following an acute intravenous injection of PLPs post-TBI, we analyzed BBB permeability, ex vivo coagulation parameters and neuroinflammation at 24h and 7d post-TBI. Both PLP-treatment and control particle-treatment had significantly decreased BBB permeability and improved clot structure 24h post-injury. Additionally, no significant change in tissue sparing was observed between 24h and 7d for PLP-treated cohorts compared to that observed in untreated cohorts. Only PLP-treatment resulted in significant reduction of astrocyte expression at 7d and percent difference from 24h to 7d. Finally, PLP-treatment significantly reduced the percent difference from 24h to 7d in microglia/macrophage density compared to the untreated control. These results suggest that PLP-treatment addressed acute hypocoagulation and decreased BBB permeability followed by decreased neuroinflammation and fold-change tissue loss by 7d post-injury. These promising results indicate that PLPs could be a potential therapeutic modality for TBI.

Introduction

With 1.7 million people affected annually and $76.5 billion USD in direct and indirect medical costs generated, traumatic brain injury (TBI) is both a leading cause of morbidity and mortality worldwide and a major burden to society ^1–3. Treating TBI patients remains challenging due to the multitude of ways the injury can occur, followed by the heterogeneous nature of the disease progression ^4,5. As the progression transverses an acute and delayed response, many pathological states can be traced to blood-brain barrier (BBB) dysfunction ⁶. One of the most problematic pathologies that occurs after TBI is coagulopathy of varying degrees. Coagulopathy increases the chances of poor clinical outcomes by greater than 30-fold relative to patients without coagulopathy ⁷. Acute coagulopathy affects more than 60% of individuals afflicted with severe TBI ^8,9 and TBI-induced coagulopathy could result in widespread intracranial hemorrhage and delayed intracranial or cerebral hematoma ^10,11.

Although coagulopathic mechanisms in TBI have not been completely understood, studies show that TBI-induced clinical coagulopathy develops rapidly from a hypercoagulable state ^12,13. Detection of fibrin degradation products and D-dimer within minutes after TBI, followed by significant reduction of fibrinogen has been reported ^12–14. Additionally, prolonged prothrombin and partial thromboplastin times are reported to occur at a late stage, indicating a transition from hyper- to a hypocoagulable state ^12–14. In this study, we focus on addressing a specific subset of coagulopathy, hypocoagulopathy, that involves disruptions to the coagulation cascade, including the dysfunction of platelets that decreases platelet-rich thrombus formation ^9,15. In addition, intracranial bleeding and platelet defects may promote high levels of neuroinflammation, mediated in part by activation of glial cells such as astrocytes and microglia ^5,9,16. Furthermore, dysregulated coagulation can cause microbleeds accompanied by the disruption of microvascular structures ^17,18 and BBB breakdown ¹⁹. Taken together, TBI-induced coagulopathy leads to changes at both acute and chronic time points in coagulation parameters, neuroinflammation and BBB permeability.

Previously, platelet transfusions have been explored as a potential therapy for TBI ⁸. However, the results of administering platelet transfusions post-TBI have been inconclusive, as hemostasis did not improve ^20,21. Therefore, there is a critical gap in understanding the therapeutic role of platelet therapy as it applies to TBI. To better understand platelet therapy as a treatment option, we have used platelet-like particles (PLPs) that have a longer shelf-life and lower immunogenicity compared to natural platelets ^22,23. The PLPs, developed by Brown et al., are comprised of an ultra-low crosslinked poly(N-isopropylacrylamide-co-acrylic-acid) microgel particles (1μm diameter), conjugated with a fibrin-specific antibody ²². These PLPs mimic platelet size, morphology and fibrin binding. By being biomimetic, biocompatible, and highly deformable, PLPs are able to mirror actions of native platelets including binding to the fibrin at injury sites, augmenting clotting and decreasing bleeding after femoral vessel injury in vivo. PLPs additionally promote clot retraction overtime, leading to increased clot density and stability ^22–26. This unique feature is due to the combination of high fibrin affinity of the fibrin-specific antibody and the high deformability of the microgel particles (Figure 1a). Previous studies by the Brown group have shown that after PLPs bind fibrin fibers within a clot, the PLPs deform and spread extensively between bound fibers. This spread confirmation is less energetically favorable than a spherical conformation, therefore the particles return to a more spherical shape. Due to the high fibrin affinity of the attached antibody, as this shape changes happens, the PLPs will deform the fibers to which they are bound, thereby inducing microcollpases within the fibrin network. These microcollapes can eventually build to create an overall bulk clot retraction. The clot retraction feature of the PLPs is a unique property that leads to increased clot density and stability which improves the overall structural and mechanical properties of clots that are formed in the presence of PLPs. Studies have also shown that this increased matrix stiffness can promote increased cell migration in vitro and enhanced healing in vivo after injury in a murine full thickness dermal injury model ²⁷. In the context of TBI, we expect that improved clot formation and stability due to PLP treatment would decrease BBB permeability and improve neuroinflammation.

Figure 1: Overview of ULC microgel-based Platelet-Like Particles.

a) i)Ultralow crosslinked (ULC) microgels are formed via a precipitation polymerization reaction of N-isopropylacrylamide with acrylic acid; ii) ULC microgels are coupled to sheep anti-human fibrin fragment E antibodies using EDC/NHS chemistry to create fibrin-targeting Platelet-Like Particles (PLPs); iii) PLPs bind and spread between several fibrin fibers within a clot, then collapse inwards to return to a non-spread, more energetically favorable conformation. This causes them to exert strain upon the bound fibrin fibers, creating microcollapses in the clot network. These microcollapses are propagated through the network via a Brownian wrench mechanism, resulting in bulk clot contraction; this results in an overall increase in clot density and stability. b) Representative AFM images and corresponding height traces (c) of ULC microgels indicate that they are able to deform significantly on glass coverslips, spreading to average diameters of 1.7 ± 0.2 μm and flattening to average heights of 15 ± 3 nm. n = 30 particles.

Although the mechanisms behind TBI-induced coagulopathy have yet to be completely elucidated, we evaluated hemostasis via the outcomes of downstream pathologies. We hypothesized that the delivery of platelet-like particles (PLPs) after TBI will combat hypocoagulation, subsequently increasing fibrin density and decreasing BBB permeability and neuroinflammation. To test our hypothesis, a mouse model of focal TBI (controlled cortical impact; CCI) was used to evaluate the effect of acute PLP-treatment compared to control ultra-low crosslinked particle treated (ULC-treated), untreated (UT), and naïve cohorts. Outcome metrics included assessment of BBB permeability at 6 hours post-injury with Evans Blue dye and fibrin density and the neuroinflammatory response at acute, 24 hours (24h) and delayed, 7 days (7d) time points via cryoSEM analysis of platelet-poor plasma and immunohistochemistry, respectively.

Materials and Methods

PLP Production

We synthesized ultralow crosslinked (ULC) poly (N-isopropylacrylamide-co-acrylic acid) (pNIPAm) microgels in a precipitation polymerization reaction as previously described ²². The reaction was initiated by adding 1 mM ammonium persulfate (APS) to a 140 mM monomer solution consisting of 90% NIPam and 10% Acrylic Acid (AAc) and allowed to continue for 6 hours at 70°C under nitrogen flux. The final product was cooled overnight and then dialyzed (Spectrum Labs, MWCO 1000 kD) against ultrapure water for 3 days with one water change per day for purification. Purified ULCs were lyophilized and then resuspended at 0.5 mg/ml for antibody conjugation. PLPs were synthesized by covalently coupling a sheep anti-human fibrin fragment E polyclonal antibody (Affinity Biologicals, Hamilton, Ontario, CA) to the AAc groups present on ULCs using EDC/NHS chemistry. PLPs were purified via dialysis (Spectrum Labs, MWCO 1000 kD) against ultrapure water for 2 days with 2 water changes per day. Purified PLPs were lyophilized, resuspended in saline, and then stored at −80 °C for future experiments.

Particle Characterization

Atomic force microscopy (AFM) (Asylum Research MFP-3D) images were obtained on coverslips cleaned through 10 minute sonication periods in solutions of 5% alconox, deionized water, acetone, absolute ethanol and isopropyl alcohol. Coverslips were then placed in a 12-well plate, treated with (3-aminopropyl)triethoxysilane for 2 hours, and washed with deionized water. ULC microgel dilutions in ultrapure water were placed over the coverslips and centrifuged for 10 minutes at 3700 rpm. ULC solutions were removed, coverslips washed with ultrapure water, and allowed to dry completely prior to imaging. Height traces for 30 particles were measured using Asylum Research software. Particle diameter for 30 particles was measured using ImageJ software.

Dynamic light scattering (DLS) (Malvern Zetasizer Nano S) was used to determine ULC microgel hydrodynamic diameter. Lyophilized ULC microgels were resuspended at 0.01 mg/mL in 10 mM HEPES buffer (pH 7.4) and vortexed to ensure uniform suspension prior to DLS measurements.

Nanosight particle tracking software (Malvern) was used to determine PLP particle concentration. CBQCA Protein Quantitation Kit (ThermoFisher) was conducted in duplicate for three PLP particle samples, using Fragment E Fibrin Antibody as a standard curve. From a known particle concentration and antibody quantification from the CBQCA assay, the amount of antibody per particle was calculated.

Animals

Male adult C57BL/6 mice (Jackson Laboratory) aged 8–10 weeks (20–24g) were used for all experiments. Mice were housed in a 14 h light/10 h dark cycle at a constant temperature (23°C ± 2° C) with food and water available ad libitum. Animal studies were approved by Arizona State University’s Institutional Animal Use and Care Committee (IACUC) and were performed in accordance with the relevant guidelines.

Controlled cortical impact model

Traumatic brain injury (TBI) was modeled using the well-established controlled cortical impact (CCI) injury model ^28–30. Briefly, anesthetized (isoflurane) adult mice were mounted onto a stereotaxic frame. The frontoparietal cortex was exposed via 3–4mm craniotomy and the impact tip was centered to the craniectomy. The impactor tip diameter was 2mm, the impact velocity was 6.0m/s and the depth of cortical deformation was 2mm and 100ms impact duration (Impact ONE; Leica Microsystems). The skin was sutured and the animals were placed in a 37°C incubator until consciousness was regained. The naïve group did not undergo anesthesia or injury procedure.

Platelet-Like Particle Injections

Intravenous injection of PLPs and control, unconjugated ULC particles was performed via retro-orbital injections of the venous sinus (for Evan’s Blue analysis cohorts) or tail-vein injections for all other cohorts. PLPs and ULCs were lyophilized and resuspended in saline for a final concentration of 2.5mg/mL. At 2–3h following the CCI, animals in PLP or ULC treatment groups were anesthetized with isoflurane (3%) and received a 100μL intravenous injection of either PLPs or ULCs 2–3h post-injury at 10mg/kg body weight. UT control cohort received no particle injection.

BBB Permeability – EB Analysis

Male mice received the CCI followed by random assigned to PLP-treatment, ULC-treatment, or untreated (UT) (n = 5–7 per group). Evan’s blue dye (EB) was dissolved in 0.9% sterile saline for a 4% w/v% and filtered via 0.22μm syringe filter (Hello Bio, Princeton, NJ, USA). At 5.5h post-injury, animals under isoflurane anesthesia (3%) received a retro-orbital injection of EB (50μL). After 30min circulation, mice were deeply anesthetized with a lethal dose of sodium pentobarbital solution. After the loss of a tail/toe pinch reflex movement, animals were transcardially perfused with cold phosphate-buffered saline (PBS) and tissue extracted immediately for analysis. The fresh brain tissue collected tissue was extracted, hemispheres were separated, and white matter was removed to focus the analysis on the cortex. A 4mm biopsy punch was used to obtain the injury region and an identical region in the contralateral hemisphere (Integra Miltex®, Princeton, NJ, USA). The remainder of the cortices were collected. All tissue was weighed and flash frozen and stored at −80°C. Thawed tissue samples were incubated in 300μL of N,N-dimethylformamide (DMF) overnight at room temperature (RT). Absorbance at 620nm and 740nm was assessed via 96-well plate absorbance reader. The linear standard curve was used to determine the concentration of dye present in each sample. Technical replicates (n = 2) were completed for each sample. Data were presented as mass of EB dye ng per g tissue.

Cohorts for Coagulation and Immunohistochemistry Analysis

Animals received the CCI injury (n=4 per cohort), and PLP and ULC were injected as described above. UT animals received no injection. At either 24h or 7d, mice were deeply anesthetized with a lethal dose of sodium pentobarbital solution. After the loss of a tail/toe pinch reflex movement, animals were transcardially perfused with cold phosphate-buffered saline (PBS) followed by 4 % buffered paraformaldehyde solution. Whole blood was obtained from animals via cardiac puncture during the transcardial perfusion with a 0.9% sodium citrate buffer washed syringe (prior to 4% PFA). Blood samples were immediately centrifuged twice at 1000 rpm for 15 mins, after which the supernatant was collected. The platelet-poor plasma (PPP) was then stored at −80 °C prior to analysis. Perfused brain tissue was collected and fixed overnight in 4 % (w/v) buffered paraformaldehyde followed by immersion in 30 % (w/v) sucrose solutions in 1X PBS for cryoprotection until the tissue was fully infiltrated. Samples were embedded in optimal cutting temperature (OCT) medium and frozen by placing in a glass container with methylbutane kept on dry ice. Samples were stored at −80°C until sectioned coronally at a 20μm thickness with a cryostat (CryostarTM NX70, ThermoFisher scientific (Waltham, MA, USA)).

Evaluation of platelet-poor plasma clot structure

Plasma clot structure was assessed for all sample groups using a JEOL 7600F cryogenic scanning electron microscope (cryoSEM) with a Gatan Alto cryo-transfer system. Additionally, wildtype BALB/c plasma was analyzed as a healthy control (Innovative research, Novi, MI, USA). 50 μl clots consisting of 90% plasma by volume were formed with 0.5 U/ml of human thrombin (Enzyme Research Laboratories, Chicago, IL, USA) and allowed to polymerize for two hours prior to imaging. Clots were rapidly frozen in sub-cooled liquid nitrogen and imaged at 2,500x. Images were taken randomly per clot (n=3 clots/cohort). ImageJ software (National Institute of Health, Bethesda, MD, USA) was utilized to create binary images and quantify fiber density determined from the ratio of black (fiber) over white (background) pixels in each image.

Immunohistochemical staining and imaging

Four tissue sections per animal (located approximately −1.65 mm Bregma with a minimum of 100 μm between sections) were analyzed following 24h and 7d post-injury (n = 4 animals per treatment/time point). Sections were stored at −20 °C until use and were brought to RT in a bath of 1X PBS. Immunohistochemistry for Iba1 and GFAP were stained simultaneously. Samples were blocked (8% horse serum (Sigma Aldrich, St. Louis, MO, USA), 0.2% Triton X-100 (ThermoFisher Scientific, Waltham, MA, USA), 1X PBS) for one hour at RT. The primary antibody solutions (anti-GFAP, chicken polyclonal (Abcam, Cambridge, MA, USA) – 1:500; anti-Iba1, rabbit polyclonal (Wako, Richmond, VA, USA) – 1:100) were incubated overnight at 4 °C. The secondary antibody solutions (donkey anti-chicken Alexa Fluor® 594 (Thomas Scientific, Swedesboro, NJ, USA) – 1:400; donkey anti-rabbit Alexa Fluor® 488 (ThermoFisher Scientific, Waltham, MA, USA) – 1:1000) were incubated for 2 hours at RT. 4,6-Diamidino-2-phenylindole (DAPI) (ThermoFisher Scientific, Waltham, MA, USA) was incubated (1 drop:1 mL of 1X PBS) on the tissue for 5 minutes at RT. Between each step, tissue was washed twice for 5 minutes each with 1XPBS. Coverslips were mounted with VectaShield fluorescent mounting media (Vector Laboratories, Burlingame, CA, USA) and sealed with nail polish.

Tissue was imaged via fluorescent microscopy (3 μm z-stack, 1.00 μm interval, 20x/0.8 objective). A refractive index of 1.49 was used to account for the VectaShield fluorescent mounting media. The Leica LASX software was used in both collection and subsequent processing (Leica Microsystems, Buffalo Grove, IL, USA). For each tissue section, the region of interest (ROI) consisted of three 20x images adjacent to the injury penumbra of the ipsilateral cortex and two 20x images from the contralateral cortex. Images acquisition settings (exposure and gain) remained constant for both 24h and 7d timepoints. Fluorescent images were deconvolved using the Blind method, auto generated point spread function, and background removed.

Immunohistochemical Image Quantification

Both GFAP and Iba1 analyses were conducted using ImageJ software. GFAP fluorescent area was calculated using the previously described protocol ³¹; briefly, the ipsilateral positive signal area was normalized by the contralateral positive signal area to account for variations in IHC signal section to section ³¹. Iba1 quantification was modified from previously described protocol ³². Cell soma counting of positive Iba1 cells (microglia/macrophage) was performed by a blinded investigator. Microglial branches/processes not associated with a cell soma were excluded from this count. The analyzed image field area was 640×480 μm, or 0.308 mm². Data were presented as total Iba1+ cell counts per image field (0.308mm² area of tissue).

The tissue sparing analysis was conducted in ImageJ software with 5x microscopy images. Three technical replicates were measured for each hemisphere with area from ventricles subtracted. The DAPI stain was used to differentiate the tissue’s edge from the background; therefore, positive tissue area was defined as tissue with positive DAPI stain. Data are presented as the ratio of tissue spared, calculated by the ipsilateral tissue area divided by the contralateral tissue area. The four tissue sections per animal (located approximately −1.65 mm Bregma with a minimum of 100 μm between sections) were averaged for each animal.

Statistical Analysis

Statistical analysis was performed in GraphPad PRISM 8.3.0 (GraphPad Software Inc., La Jolla, CA, USA). Fiber density was assessed via ordinary one-way ANOVA per timepoint, followed by Tukey’s post-hoc test. For all other statistical analyses, an ordinary, two-way ANOVA was conducted to analyze UT and PLP-treated cohorts individually. If there was a significant main effect of treatment (UT vs. PLP-treated) and/or significant main effect of timepoint (24h vs. 7d), the two-way ANOVA was followed by a post-hoc Bonferroni’s multiple comparison test. For percent difference analysis, an unpaired, two-tailed Student’s t-test was conducted. Grubb’s test was used to identify all outliers.

Results

Particle Characteristics

ULC microgels were characterized by DLS to determine size in solution; DLS showed microgel diameters to be 1.56 μm ± 97.4 nm. Particles were also characterized via AFM imaging to determine size and deformability by analyzing particle spreading on a glass slide. AFM imaging revealed average diameters of 1.7 μm ± 0.2 μm and heights of 15 ± 3 nm (Figure 1b–c). High level of spreading and low heights are indicative of high levels of particle deformability. Finally, antibody conjugation per particle was determined to be 2.04 X 10^5 ± 1.61 X 10^4 Fragment E fibrin antibody molecules per microgel particle. The ULC particles Nanosight particle tracking for particle concentration shows there are 1.02 X 10^9 particles/mL in a 0.05mg/mL solution of PLPs. With this known particle concentration, a CBQCA assay with Fragment E fibrin antibody as standard curve was run and measured an average of 2.04 × 10^5 ± 1.61 × 10^4 Fragment E fibrin antibody molecules per PLP particle.

Decreased BBB Permeability Following PLP-Treatment

BBB permeability post-TBI was measured via EB exogenous dye for naïve, UT, ULC-treated and PLP-treated animals. EB was injected (4% w/v) at 5.5h post-injury, and allowed to circulate for 30 minutes before sacrifice. This experimental paradigm allowed for an acute assessment of the compromised BBB within a previously determined window of permeability ^33,34. A two-way ANOVA indicated significant interactions in both hemisphere and treatment. Only UT animals had a significantly greater amount of EB present in the ipsilateral core (80.83 ± 58.70 ng EB per g tissue) when compared to the contralateral core (5.61 ± 3.27 ng EB per g, p<0.0001) (Figure 2). The UT ipsilateral core also demonstrated a significantly greater amount of EB extravasation compared to the naïve cortical tissue (3.57 ± 2.65 ng EB per g, p=0.0002), ULC-treated ipsilateral core (26.82 ± 12.56 ng EB per g, p=0.0070), and PLP-treated ipsilateral core (23.69 ± 9.10 ng EB per g, p=0.0039). Complete data and analyses of the ipsilateral and contralateral hemispheres outside of the injury core are presented in Supplemental Figure 1.

Figure 2: Decreased BBB Permeability Following Platelet-Like Particle Treatment.

EB was evaluated via absorbance assay, and the calculated mass (ng) was normalized by the respective tissue biopsy punch (g) (n=5–7 per cohort). Significant increase in EB extravasation in UT ipsilateral injury core. Mean ± SEM, ** p<0.01, *** p<0.001, **** p<0.0001.

Platelet-Like Particles Rescue Clot Structure

Cryo-SEM analysis of clots formed from PPP indicated that UT animals had significantly disrupted clot structure at 24h, marked by a high level of porosity and low fiber density compared to naïve controls (representative images in Figure 3a). At 24h post-injury, clots formed from PLP-treated animals had significantly greater fiber density (0.81 ± 0.12) than ULC-treated (0.17 ± 0.04, p=0.0007) or UT animals (0.23 ± 0.11, p=0.0004) (Figure 3b). At 7d post-injury, clot structure was similar among PLP, ULC, and UT cohorts (0.23 ± 0.44, 0.23 ± 0.04, 0.22 ± 0.08, respectively; Figure 3c); however, clots formed from all injured mice regardless of intervention all exhibited significantly lower fiber density compared to clots formed from naïve animals (0.93 ± 0.13).

Figure 3: Cryo-SEM images of mouse fibrin networks formed from platelet-poor plasma collected 24h or 7d post TBI and treatment with platelet-like particles.

Clots were polymerized for 2 hours prior to imaging at 2,500x. Scale bar= 10 μm. a) Representative images from normal/naive clots are compared to UT, ULC, and PLP treatments at 24h and 7d. b) Fiber density (black/white pixels) was calculated for 24h post-injury. c) Fiber density (black/white pixels) was calculated for 7d post-injury. Mean ± SEM, n=3/group, *** p<0.001.

Platelet-Like Particle Driven Reduction in Reactive Astrocytes

Reactive astrocytes levels were measured by immunostaining for GFAP at 24h and 7d post-injury. Due to the significant improvement in fibrin density in PLP-treated animals, we chose to focus solely on this treatment in comparison to UT animals. Data were analyzed and presented by the percent GFAP+ area in cortical tissue adjacent to injury penumbra normalized to contralateral cortical GFAP+ area. Representative images illustrate the PLP-driven reduction in reactive astrocytes within the injury penumbra (Figure 4a). A two-way ANOVA showed significant effect due to timepoint. Specifically, significance was observed at 7d between PLP-treated (5.74 ± 2.61) and UT cohorts (10.77 ± 3.73, p=0.0280), and a significant increase in GFAP+ area from 24h to 7d for UT animals (0.967 ± 0.25 to 10.77 ± 3.73, p=0.005, Figure 4b). Note, Grubb’s test identified an outlier in the UT 24h cohort, resulting in n=3 and n=4 for this cohort.

Figure 4: PLP driven reduction in reactive astrocytes (GFAP+).

a) Qualitative depiction of GFAP+ astrocytes within the injury penumbra (20x magnification) for both 24h and 7d in UT and PLP-treated tissue, scale bar is 100 μm. b) Normalized (ipsilateral:contralateral) GFAP+ area of UT and PLP-treated at both 24h and 7d (n=3–4). Mean ± SEM, * p<0.05, *** p<0.001

Temporal Reduction of Microglia/Macrophage per Field

Microglia and macrophage populations were assessed via immunostaining for Iba1 at 24h and 7d post-TBI. The Iba1+ cell somas were blindly counted; representative images are depicted in Figure 4a. The recorded cell count from ipsilateral regions (n=4 per cohort) were analyzed via two-way ANOVA for 24h and 7d (Figure 5b). A two-way ANOVA identified significance due to both treatment and timepoint. Notably, at 7d, UT (158.58 ± 20.74) had significantly greater microglia/macrophage presence than PLP-treated (113.08 ± 28.84, p=0.0081). Both UT and PLP-treated cohorts demonstrated a significant increase in Iba1+ cell somas present in the cortical injury penumbra from 24h (37.85 ± 4.82, 42.44 ± 5.95, respectively) to 7d (158.58 ± 20.74, 113.08 ± 28.84; p<0.001 and p=0.003, respectively). Further analysis including Iba1+ cells in the contralateral hemisphere are presented in Supplemental Figure 2.

Figure 5: Temporal reduction of macrophage/microglia per field.

a) Qualitative depiction of Iba1+ microglia/macrophage at 20x magnification for both 24h and 7d in UT and PLP-treated tissue, scale bar is 100μm. b) Comparison of ipsilateral regions of both UT and PLP-treated tissue at 24h and 7d (n=4/cohort). Mean ± SEM, ** p<0.01, *** p<0.001, **** p<0.0001.

Tissue Spared after TBI

Ratio of tissue spared after TBI was determined by normalizing the total area of ipsilateral tissue by the total contralateral tissue area as determined from DAPI stain/brightfield. Representative images of the ipsilateral hemispheres at both 24h and 7d illustrate and highlight the region of tissue loss in the ipsilateral hemisphere (Figure 6a). A two-way ANOVA identified significance due to both time and the interaction between time and treatment. Specifically, post-hoc pairwise comparisons revealed a significant decrease in tissue spared in UT cohorts from 24h (0.95 ± 0.01) to 7d (0.89 ± 0.03; p=0.0056, Figure 6b). The subsequent percent change analysis further demonstrated the significant decrease in the UT cohorts between 24h and 7d when compared to the PLP-treated cohorts ((p=0.030; t-test, Figure 6c).

Figure 6: Tissue Spared after TBI.

a) Qualitative images (5x, DAPI, blue) of the injured ipsilateral cortex at 24h and 7d post-injury, scale bar is 1.00 mm. Gray shading highlights cortical tissue loss. b) Tissue spared (ipsi/contra) at 24h and 7d for UT and PLP-treated cohorts (n=4 per cohort). c) Percent change of tissue spared from 24hr to 7d for UT and PLP-treated cohorts. Mean ± SEM, * p<0.05, ** p<0.01.

Discussion

The objective of this study was to assess the role of coagulopathy in TBI. The previously developed PLP technology enabled unique control over coagulation targeting mechanism that is not possible with blood-derived products (i.e., platelet infusions). We hypothesized that a single, intravenous injection of PLPs acutely post-injury would improve hemostasis and thereby diminish subsequent neural and coagulation pathologies (i.e., clot dysfunction, BBB permeability, and neuroinflammation). Our BBB permeability assessment at 6h post-injury revealed marked BBB permeability in the CCI model when no intervention was provided. However, both the PLP and ULC-treated animals demonstrated significant reduction in BBB permeability. Evaluating the clot function at 24h post-injury, we observed a significant increase in fibrin density only in the PLP-treated cohorts as compared to the UT and ULC control cohorts. Moreover, we observed a significant decrease in the astrocyte reactivity and microglial expression with the PLP treatment compared to untreated controls, suggesting an overall decrease in neuroinflammation. The impact of PLP treatment was further supported by the tissue sparing analysis whereby the PLP-treated cohort did not exhibit further tissue loss from 24h to 7d as observed in the untreated cohorts.

TBI results in a disease progression whereby pathophysiological events evolve from the acute to the delayed response ¹⁹. Acutely within minutes to hours, compromised blood vessels largely contribute to BBB permeability and dysfunction ³⁴. Our data supported this notion as we observed marked extravasation of EB dye in TBI mice at 6h post-injury. We and others have further characterized the temporal profile for BBB permeability in the CCI model whereby BBB permeability is heightened in the first 12h post-CCI including extravasation of macromolecules ^34–36 and nanoparticles ^31,33. This extravasation was significantly inhibited when treated with PLPs or ULCs. These results suggest that the PLPs and the control ULC system impact injury-induced BBB permeability within 6hrs after injury. The action of the control ULCs may be due to simple colloidal effects limiting EB dye extravasation within the short experimental time frame; however, we cannot rule out additional factors such as altered systemic inflammatory/immune cell activity as observed in other traumatic injury model with intravenous particle administration (spinal cord injury) ³⁷. Notably, evaluation of clot structure 24h post-injury/intervention revealed a significant effect of only the PLPs to stabilize the coagulation from the injury induced hypocoagulation state. This effect was transient and was not observed at the 7d time-point, regardless of intervention. These results demonstrate the PLPs’ specificity of the anti-fibrin influence coagulation on demand.

TBI-induced coagulopathy may lead to BBB dysfunction and permeability and may contribute to neuroinflammation, including extravasation and deposition of fibrin(ogen) in and of itself ^{17,19,38–42}. Here, we evaluated alterations in both astrocytes and microglia, which have different temporal profiles for neuroinflammatory expression ^43,44. Reactive astrocyte expression is often reported in the injury penumbra beginning three days post injury ⁴⁴. No significant change was noted in GFAP expression in the PLP-treatment compared to UT controls at 24h. However, at 7d post-injury, a significant increase in GFAP expression for the UT animals, but not SP treated was observed, consistent with the previously established neuroinflammatory timeline for astrocytes ⁴⁴. Macrophage and microglia cell density typically begins to emerge acutely (2–3 hours) post-injury and continue migrating to the injury region over the course of a week post-injury ^32,44,45. Additionally, while it cannot be implied that the PLPs decreased the presence of microglia/macrophage by 7d (due to the significant increase in Iba1 expression from 24h to 7d in both PLP-treated and UT cohorts), the differential change within the injury penumbra from 24h to 7d less for PLP-treated compared to UT cohorts. The impact of chronic and sustained activation of astrocytes and microglia contribute to long-term damage post-TBI, including glial scarring and obstruction of axon regeneration (astrocytes), and the production of pro-inflammatory and cytotoxic factors from dysregulated microglia ^45–48.

While necrosis begins at the initial insult, TBI results in both immediate and delayed cell death that can continue for years ^49,50. Here, we assessed macroscopic tissue loss due to necrosis, apoptosis, and/or necroptosis through tissue sparing measurements. In this study, we observed no significant difference between UT- and PLP-treated cohorts at either 24h or 7d. However, there was a significant decrease in tissue spared from 24h to 7d for the UT cohorts, whereas no significance was displayed for PLP-treated cohorts. Since inflammation can contribute to changes in lesion size, the lack of significant decrease in tissue spared by 7d suggests that PLPs reduce inflammation, and potentially inhibit the tissue loss progression. Prior studies in an ischemic stroke model with platelet-rich plasma greatly enhanced tissue sparing compared to non-treated control stroke animals ⁵¹. While direct comparisons between our study and this prior work is difficult, it suggests that the intervention paradigm used in our study may be sub-optimal (i.e., adjust concentration and number of PLPs injections). Additionally, PLPs lack the growth factors present in platelet-rich-plasma which could also add to these differences and could motivate the loading of growth factors into PLPs in future studies.

In this study, we used a clinically relevant and translational approach for SP delivery. A single intravenous injection of PLPs at acute time point significantly impacted key aspects of subsequent TBI pathologies within 7d post-injury. Thus, signifying the importance of an early intervention for TBI therapeutics. To the best of our knowledge, ours is the first study to demonstrate that SP has a protective effect on coagulation, neuroinflammation and BBB disruption after TBI in mice. As previously mentioned, the exact mechanisms surrounding TBI-induced coagulopathy have yet to be fully understood. Many hypotheses of these mechanisms point to dysregulated amplification of tissue factor expression or depletion of the α−2 plasmin inhibitor ^52–55. The commonality between these hypotheses is the progression to fibrinolysis. However, the unique deformability of the PLPs has been shown to combat fibrinolysis through increasing the clot density ^22,24. Therefore, it is possible that the PLPs had the same effect in a TBI model. For this reason, future studies should include evaluation of fibrinolysis rates post-TBI.

Delivery of hemostatic therapies, such as platelet transfusions, have been investigated in a clinical setting ^21,56,57. However, these results have been either inconclusive or ineffective. The PLPs are unique in that they promote hemostasis even in highly coagulopathic conditions ^22,26,27. Natural platelet transfusions may be ineffective due to the inherent dysfunction of the coagulation cascade post-TBI. Thus, the independence of the PLPs from a robust coagulation cascade allows for improved hemostasis thereby influencing downstream pathologies such as neuroinflammation. Furthermore, the transition from hypo- to hypercoagulation state and the dynamics of the BBB breakdown play a critical role in determining the therapeutic window for SP delivery. In this study, the acute time point for single SP injection at 2h post-TBI was chosen based on previous literature that suggests early platelet activation may improve recovery ^51,58. In addition, we have previously demonstrated that focal TBI leads to peak BBB disruption at 1–3h post-injury ^31,33. However, future studies investigating the alterations in the timing of administration and/or if multiple injections would improve therapeutic outcomes are warranted.

In conclusion, the PLPs provided promising results with respect to both neuroinflammation and tissue sparing with only one injection. Acute administration of PLPs post-injury may have allowed the PLPs to stabilize the fibrin matrices early enough to prevent complete progression of sub-acute pathologies, such as neuroinflammation. Although TBI-induced coagulopathic mechanisms are yet to be clearly elucidated, PLPs evidently demonstrate to significantly improve the downstream pathologies. Thus, our promising results show that PLPs could be a potential therapeutic modality for TBI.