Arginine vasopressin receptor 2 activation promotes microvascular permeability in sepsis.

Methicillin-resistant Staphylococcus aureus (MRSA) sepsis is a severe condition associated with vascular leakage and poor prognosis. The hemodynamic management of sepsis targets hypotension, but there is no specific treatment available for vascular leakage. Arginine vasopressin (AVP) has been used in sepsis to promote vasoconstriction by activating AVP receptor 1 (V1R). However, recent evidence suggests that increased fluid retention may be associated with the AVP receptor 2 (V2R) activation worsening the outcome of sepsis. Hence, we hypothesized that the inhibition of V2R activation ameliorates the severity of microvascular hyperpermeability during sepsis. The hypothesis was tested using a well-characterized and clinically relevant ovine model of MRSA pneumonia/sepsis and in vitro assays of human lung microvascular endothelial cells (HMVECs). in vivo experiments demonstrated that the treatment of septic sheep with tolvaptan (TLVP), an FDA-approved V2R antagonist, significantly attenuated the sepsis-induced fluid retention and markedly reduced the lung water content. These pathological changes were not affected by the treatment with V2R agonist, desmopressin (DDAVP). Additionally, the incubation of cultured HMVECs with DDAVP, and DDAVP along with MRSA significantly increased the paracellular permeability. Finally, both the DDAVP and MRSA-induced hyperpermeability was significantly attenuated by TLVP. Subsequent protein and gene expression assays determined that the V2R-induced increase in permeability is mediated by phospholipase C beta (PLCß) and the potent permeability factor angiopoietin-2. In conclusion, our results indicate that the activation of the AVP-V2R axis is critical in the pathophysiology of severe microvascular hyperpermeability during Gram-positive sepsis. The use of the antagonist TLVP should be considered as adjuvant treatment for septic patients. The results from this clinically relevant animal study are highly translational to clinical practice.

Blood carboxyhemoglobin elimination curve, half-lifetime, and arterial-venous differences in acute phase of carbon monoxide poisoning in ovine smoke inhalation injury model.

Smoke inhalation injury (SII) affects more than 50,000 people annually causing carbon monoxide (CO) poisoning. Although the increased blood level of carboxyhemoglobin (CO-Hb) is frequently used to confirm the diagnosis of SII, knowledge of its elimination in the acute phase is still limited. The aim of this study is to determine CO-Hb elimination rates and their differences in arterial (aCO-Hb) and mixed-venous (vCO-Hb) blood following severe SII in a clinically relevant ovine model. Forty-three chronically instrumented female sheep were subjected to SII (12 breaths, 4 sets) through tracheostomy tube under anesthesia and analgesia. After the SII, sheep were awakened and placed on a mechanical ventilator (FiO2 = 1.0, tidal volume 12 mL/kg, and PEEP = 5cmH2O) and monitored. Arterial and mixed-venous blood samples were withdrawn simultaneously for blood gas analysis at various time points to determine CO-HB half-lifetime and an elimination curve. The mean of highest aCO-Hb level during SII was 70.8 ± 13.9%. The aCO-Hb elimination curve showed an approximated exponential decay during the first 60 min. Per mixed linear regression model analysis, aCO-Hb significantly (p < 0.001) declined (4.3%/minute) with a decay constant lambda of 0.044. With this lambda, mean lifetime and half-lifetime of aCO-Hb were 22.7 and 15.7 min, respectively. The aCO-Hb was significantly lower compared to vCO-Hb at all-time points (0-180 min). To our knowledge, this is the first report describing CO-Hb elimination curve in the acute phase after severe SII in the clinically relevant ovine model. Our data shows that CO-Hb is decreasing in linear manner with supportive mechanical ventilation (0-60 min). The results may help to understand CO-Hb elimination curve in the acute phase and improvement of pre-hospital and initial clinical care in patients with CO poisoning.

Surgical anatomy of ovine facial and hypoglossal nerves for facial nerve reconstruction and regeneration research: An experimental study in sheep.

BACKGROUND: The lack of a clinically relevant animal model for facial nerve research is a challenge. The goal of this study was to investigate the anatomy of the ovine facial and hypoglossal nerves to establish a clinically relevant facial nerve research model. MATERIALS AND METHODS: Six cadaver female Merino sheep (33.5 ± 3 kg, approximately 3 years old) and three anesthetized female Merino sheep (30 ± 3 kg, approximately 3 years old) were used. In cadaver sheep, a right side preauricular to submandibular incision was made. Dimensions of the face, neck, and length of facial nerve were measured. In anesthetized sheep, each facial nerve branch and hypoglossal nerve in the right side was stimulated. The number of myelinated fibers was analyzed histologically. RESULTS: The facial nerve exited the stylomastoid foramen and divided into upper and lower branches. The lower branch then subdivided into buccal and marginal mandibular branches. The hypoglossal nerve was observed behind the digastric posterior belly. Stimulation revealed the temporal, zygomatic, buccal, marginal mandibular, and cervical branch innervated the forehead, orbicularis, upper lip and nasal, lower lip, and platysma, respectively. The number of myelinated fibers of the main trunk, upper, buccal, lower branch, and hypoglossal nerve was 11 350 ± 1851, 4766 ± 1000, 5107 ± 218, 3159 ± 450, and 7604 ± 636, respectively. The length of the main trunk was 9.2 ± 1.5 mm, and distance of the marginal mandibular branch to the facial artery was 94 ± 6.8 mm. CONCLUSIONS: Due to the similarity in nerve anatomy and innervation, the ovine model can be used as a clinically relevant and suitable model for facial nerve research.

Population Kinetics of 0.9% Saline Distribution in Hemorrhaged Awake and Isoflurane-anesthetized Volunteers.

BACKGROUND: Population-based, pharmacokinetic modeling can be used to describe variability in fluid distribution and dilution between individuals and across populations. The authors hypothesized that dilution produced by crystalloid infusion after hemorrhage would be larger in anesthetized than in awake subjects and that population kinetic modeling would identify differences in covariates. METHODS: Twelve healthy volunteers, seven females and five males, mean age 28 ± 4.3 yr, underwent a randomized crossover study. Each subject participated in two separate sessions, separated by four weeks, in which they were assigned to an awake or an anesthetized arm. After a baseline period, hemorrhage (7 ml/kg during 20 min) was induced, immediately followed by a 25 ml/kg infusion during 20 min of 0.9% saline. Hemoglobin concentrations, sampled every 5 min for 60 min then every 10 min for an additional 120 min, were used for population kinetic modeling. Covariates, including body weight, sex, and study arm (awake or anesthetized), were tested in the model building. The change in dilution was studied by analyzing area under the curve and maximum plasma dilution. RESULTS: Anesthetized subjects had larger plasma dilution than awake subjects. The analysis showed that females increased area under the curve and maximum plasma dilution by 17% (with 95% CI, 1.08 to 1.38 and 1.07 to 1.39) compared with men, and study arm (anesthetized increased area under the curve by 99% [0.88 to 2.45] and maximum plasma dilution by 35% [0.71 to 1.63]) impacted the plasma dilution whereas a 10-kg increase of body weight resulted in a small change (less than1% [0.93 to 1.20]) in area under the curve and maximum plasma dilution. Mean arterial pressure was lower in subjects while anesthetized (P < 0.001). CONCLUSIONS: In awake and anesthetized subjects subjected to controlled hemorrhage, plasma dilution increased with anesthesia, female sex, and lower body weight. Neither study arm nor body weight impact on area under the curve or maximum plasma dilution were statistically significant and therefore no effect can be established.

Mild, moderate and severe: terminology implications for clinical and experimental traumatic brain injury.

PURPOSE OF REVIEW: When describing clinical or experimental traumatic brain injury (TBI), the adjectives 'mild,' 'moderate' and 'severe' are misleading. 'Mild' clinical TBI frequently results in long-term disability. 'Severe' rodent TBI actually resembles mild or complicated mild clinical TBI. RECENT FINDINGS: Many mild TBI patients appear to have recovered completely but have postconcussive symptoms, deficits in cognitive and executive function and reduced cerebral blood flow. After moderate TBI, 31.8% of patients died or were discharged to skilled nursing or hospice. Among survivors of moderate and severe TBI, 44% were unable to return to work. On MRI, 88% of mild TBI patients have evidence of white matter damage, based on measurements of fractional anisotropy and mean diffusivity/apparent diffusion coefficient. After sports concussion, clinically recovered patients have abnormalities in functional connectivity on functional MRI. Methylphenidate improved fatigue and cognitive impairment and, combined with cognitive rehabilitation, improved memory and executive functioning. In comparison to clinical TB, because the entire spectrum of experimental rodent TBI, although defined as moderate or severe, more closely resembles mild or complicated mild clinical TBI. SUMMARY: Many patients after mild or moderate TBI suffer long-term sequelae and should be considered a major target for translational research. Treatments that improve outcome in rodent TBI, even when the experimental injuries are defined as severe, might be most applicable to mild or moderate TBI.

Proteomic changes in traumatic brain injury: experimental approaches.

PURPOSE OF REVIEW: The underlying mechanisms responsible for chronic and progressive neurological damage after traumatic brain injury (TBI) are poorly understood, and therefore, current treatment options are limited. Proteomics is an emerging methodology to study changes to the TBI proteome in both patients and experimental models. RECENT FINDINGS: Although experimentally complex, mass spectrometry-based proteomics approaches are converging on a set of common methods. However, these methods are being applied to an increasingly diverse range of experimental models and types of injury. SUMMARY: In this review, our aim is to briefly describe experimental TBI models and the underlying methods common to most proteomic approaches. We will then review a series of articles that have recently appeared in which these approaches have been applied to important TBI questions. We will summarize several recent experimental studies, and suggest how the results of these emerging studies might impact future research as well as patient treatment.

Impact & Blast Traumatic Brain Injury: Implications for Therapy.

Traumatic brain injury (TBI) is one of the most frequent causes of combat casualties in Operations Iraqi Freedom (OIF), Enduring Freedom (OEF), and New Dawn (OND). Although less common than combat-related blast exposure, there have been significant numbers of blast injuries in civilian populations in the United States. Current United States Department of Defense (DoD) ICD-9 derived diagnoses of TBI in the DoD Health Care System show that, for 2016, severe and moderate TBIs accounted for just 0.7% and 12.9%, respectively, of the total of 13,634 brain injuries, while mild TBIs (mTBIs) accounted for 86% of the total. Although there is a report that there are differences in the frequency of long-term complications in mTBI between blast and non-blast TBIs, clinical presentation is classified by severity score rather than mechanism because severity scoring is associated with prognosis in clinical practice. Blast TBI (bTBI) is unique in its pathology and mechanism, but there is no treatment specific for bTBIs-these patients are treated similarly to TBIs in general and therapy is tailored on an individual basis. Currently there is no neuroprotective drug recommended by the clinical guidelines based on evidence.

Nebulized Epinephrine Limits Pulmonary Vascular Hyperpermeability to Water and Protein in Ovine With Burn and Smoke Inhalation Injury.

OBJECTIVES: To test the hypothesis that nebulized epinephrine ameliorates pulmonary dysfunction by dual action-bronchodilation (ß2-adrenergic receptor agonism) and attenuation of airway hyperemia (α1-adrenergic receptor agonism) with minimal systemic effects. DESIGN: Randomized, controlled, prospective, and large animal translational studies. SETTING: University large animal ICU. SUBJECTS: Twelve chronically instrumented sheep. INTERVENTIONS: The animals were exposed to 40% total body surface area third degree skin flame burn and 48 breaths of cooled cotton smoke inhalation under deep anesthesia and analgesia. The animals were then placed on a mechanical ventilator, fluid resuscitated, and monitored for 48 hours in a conscious state. After the injury, sheep were randomized into two groups: 1) epinephrine, nebulized with 4 mg of epinephrine every 4 hours starting 1 hour post injury, n = 6; or 2) saline, nebulized with saline in the same manner, n = 6. MEASUREMENTS AND MAIN RESULTS: Treatment with epinephrine had a significant reduction of the pulmonary transvascular fluid flux to water (p < 0.001) and protein (p < 0.05) when compared with saline treatment from 12 to 48 hours and 36 to 48 hours, respectively. Treatment with epinephrine also reduced the systemic accumulation of body fluids (p < 0.001) with a mean of 1,410 ± 560 mL at 48 hours compared with 3,284 ± 422 mL of the saline group. Hemoglobin levels were comparable between the groups. Changes in respiratory system dynamic compliance, mean airway pressure, PaO2/FiO2 ratio, and oxygenation index were also attenuated with epinephrine treatment. No considerable systemic effects were observed with epinephrine treatment. CONCLUSIONS: Nebulized epinephrine should be considered for use in future clinical studies of patients with burns and smoke inhalation injury.

Measurement of Postreplicative DNA Metabolism and Damage in the Rodent Brain.

The DNA of all organisms is metabolically active due to persistent endogenous DNA damage, repair, and enzyme-mediated base modification pathways important for epigenetic reprogramming and antibody diversity. The free bases released from DNA either spontaneously or by base excision repair pathways constitute DNA metabolites in living tissues. In this study, we have synthesized and characterized the stable-isotope standards for a series of pyrimidines derived from the normal DNA bases by oxidation and deamination. We have used these standards to measure free bases in small molecule extracts from rat brain. Free bases are observed in extracts, consistent with both endogenous DNA damage and 5-methylcytosine demethylation pathways. The most abundant free base observed is uracil, and the potential sources of uracil are discussed. The free bases measured in tissue extracts constitute the end product of DNA metabolism and could be used to reveal metabolic disturbances in human disease.

Rapid accumulation of endogenous tau oligomers in a rat model of traumatic brain injury: possible link between traumatic brain injury and sporadic tauopathies.

Traumatic brain injury (TBI) is a serious problem that affects millions of people in the United States alone. Multiple concussions or even a single moderate to severe TBI can also predispose individuals to develop a pathologically distinct form of tauopathy-related dementia at an early age. No effective treatments are currently available for TBI or TBI-related dementia; moreover, only recently has insight been gained regarding the mechanisms behind their connection. Here, we used antibodies to detect oligomeric and phosphorylated Tau proteins in a non-transgenic rodent model of parasagittal fluid percussion injury. Oligomeric and phosphorylated Tau proteins were detected 4 and 24 h and 2 weeks post-TBI in injured, but not sham control rats. These findings suggest that diagnostic tools and therapeutics that target only toxic forms of Tau may provide earlier detection and safe, more effective treatments for tauopathies associated with repetitive neurotrauma.

Human mesenchymal stem cells reduce the severity of acute lung injury in a sheep model of bacterial pneumonia.

BACKGROUND: Human bone marrow-derived mesenchymal stem (stromal) cells (hMSCs) improve survival in mouse models of acute respiratory distress syndrome (ARDS) and reduce pulmonary oedema in a perfused human lung preparation injured with Escherichia coli bacteria. We hypothesised that clinical grade hMSCs would reduce the severity of acute lung injury (ALI) and would be safe in a sheep model of ARDS. METHODS: Adult sheep (30-40 kg) were surgically prepared. After 5 days of recovery, ALI was induced with cotton smoke insufflation, followed by instillation of live Pseudomonas aeruginosa (2.5×10(11) CFU) into both lungs under isoflurane anaesthesia. Following the injury, sheep were ventilated, resuscitated with lactated Ringer's solution and studied for 24 h. The sheep were randomly allocated to receive one of the following treatments intravenously over 1 h in one of the following groups: (1) control, PlasmaLyte A, n=8; (2) lower dose hMSCs, 5×10(6) hMSCs/kg, n=7; and (3) higher-dose hMSCs, 10×10(6) hMSCs/kg, n=4. RESULTS: By 24 h, the PaO2/FiO2 ratio was significantly improved in both hMSC treatment groups compared with the control group (control group: PaO2/FiO2 of 97±15 mm Hg; lower dose: 288±55 mm Hg (p=0.003); higher dose: 327±2 mm Hg (p=0.003)). The median lung water content was lower in the higher-dose hMSC-treated group compared with the control group (higher dose: 5.0 g wet/g dry [IQR 4.9-5.8] vs control: 6.7 g wet/g dry [IQR 6.4-7.5] (p=0.01)). The hMSCs had no adverse effects. CONCLUSIONS: Human MSCs were well tolerated and improved oxygenation and decreased pulmonary oedema in a sheep model of severe ARDS. TRAIL REGISTRATION NUMBER: NCT01775774 for Phase 1. NCT02097641 for Phase 2.

Antithrombin attenuates vascular leakage via inhibiting neutrophil activation in acute lung injury.

OBJECTIVE: To test the hypothesis that restoration of antithrombin plasma concentrations attenuates vascular leakage by inhibiting neutrophil activation through syndecan-4 receptor inhibition in an established ovine model of acute lung injury. DESIGN: Randomized controlled laboratory experiment. SETTING: University animal research facility. SUBJECTS: Eighteen chronically instrumented sheep. INTERVENTIONS: Following combined burn and smoke inhalation injury (40% of total body surface area, third-degree flame burn; 4 × 12 breaths of cold cotton smoke), chronically instrumented sheep were randomly assigned to receive an IV infusion of 6 IU/kg/hr recombinant human antithrombin III or normal saline (n = 6 each) during the 48-hour study period. In addition, six sham animals (not injured, continuous infusion of vehicle) were used to obtain reference values for histological and immunohistochemical analyses. MEASUREMENTS AND MAIN RESULTS: Compared to control animals, recombinant human antithrombin III reduced the number of neutrophils per hour in the pulmonary lymph (p < 0.01 at 24 and 48 hr), alveolar neutrophil infiltration (p = 0.04), and pulmonary myeloperoxidase activity (p = 0.026). Flow cytometric analysis revealed a significant reduction of syndecan-4-positive neutrophils (p = 0.002 vs control at 24 hr). Treatment with recombinant human antithrombin III resulted in a reduction of pulmonary nitrosative stress (p = 0.002), airway obstruction (bronchi: p = 0.001, bronchioli: p = 0.013), parenchymal edema (p = 0.044), and lung bloodless wet-to-dry-weight ratio (p = 0.015). Clinically, recombinant human antithrombin III attenuated the increased pulmonary transvascular fluid flux (12-48 hr: p ≤ 0.001 vs control each) and the deteriorated pulmonary gas exchange (12-48 hr: p < 0.05 vs control each) without increasing the risk of bleeding. CONCLUSIONS: The present study provides evidence for the interaction between antithrombin and neutrophils in vivo, its pathophysiological role in vascular leakage, and the therapeutic potential of recombinant human antithrombin III in a large animal model of acute lung injury.

Mesenchymal stem cell-derived exosomes for treatment of sepsis.

Introduction: The pathogenesis of sepsis is an imbalance between pro-inflammatory and anti-inflammatory responses. At the onset of sepsis, the lungs are severely affected, and the injury progresses to acute respiratory distress syndrome (ARDS), with a mortality rate of up to 40%. Currently, there is no effective treatment for sepsis. Cellular therapies using mesenchymal stem cells (MSCs) have been initiated in clinical trials for both ARDS and sepsis based on a wealth of pre-clinical data. However, there remains concern that MSCs may pose a tumor risk when administered to patients. Recent pre-clinical studies have demonstrated the beneficial effects of MSC-derived extracellular vesicles (EVs) for the treatment of acute lung injury (ALI) and sepsis. Methods: After recovery of initial surgical preparation, pneumonia/sepsis was induced in 14 adult female sheep by the instillation of Pseudomonas aeruginosa (~1.0×1011 CFU) into the lungs by bronchoscope under anesthesia and analgesia. After the injury, sheep were mechanically ventilated and continuously monitored for 24 h in a conscious state in an ICU setting. After the injury, sheep were randomly allocated into two groups: Control, septic sheep treated with vehicle, n=7; and Treatment, septic sheep treated with MSC-EVs, n=7. MSC-EVs infusions (4ml) were given intravenously one hour after the injury. Results: The infusion of MSCs-EVs was well tolerated without adverse events. PaO2/FiO2 ratio in the treatment group tended to be higher than the control from 6 to 21 h after the lung injury, with no significant differences between the groups. No significant differences were found between the two groups in other pulmonary functions. Although vasopressor requirement in the treatment group tended to be lower than in the control, the net fluid balance was similarly increased in both groups as the severity of sepsis progressed. The variables reflecting microvascular hyperpermeability were comparable in both groups. Conclusion: We have previously demonstrated the beneficial effects of bone marrow-derived MSCs (10×106 cells/kg) in the same model of sepsis. However, despite some improvement in pulmonary gas exchange, the present study demonstrated that EVs isolated from the same amount of bone marrow-derived MSCs failed to attenuate the severity of multiorgan dysfunctions.

Effects of nebulized adipose-derived mesenchymal stem cells on acute lung injury following smoke inhalation in sheep.

INTRODUCTION: Treatment of ARDS caused by smoke inhalation is challenging with no specific therapies available. The aim of this study was to test the efficacy of nebulized adipose-derived mesenchymal stem cells (ASCs) in a well-characterized, clinically relevant ovine model of smoke inhalation injury. MATERIAL AND METHODS: Fourteen female Merino sheep were surgically instrumented 5-7 days prior to study. After induction of acute lung injury (ALI) by cooled cotton smoke insufflation into the lungs (under anesthesia and analgesia), sheep were placed on a mechanical ventilator for 48 hrs and monitored for cardiopulmonary hemodynamics in a conscious state. ASCs were isolated from ovine adipose tissue. Sheep were randomly allocated to two groups after smoke injury: 1) ASCs group (n = 6): 10 million ASCs were nebulized into the airway at 1 hr post-injury; and 2) Control group (n = 8): Nebulized with saline into the airways at 1 hr post-injury. ASCs were labeled with green fluorescent protein (GFP) to trace cells within the lung. ASCs viability was determined in bronchoalveolar lavage fluid (BALF). RESULTS: PaO2/FiO2 in the ASCs group was significantly higher than in the control group (p = 0.001) at 24 hrs. Oxygenation index: (mean airway pressure × FiO2/PaO2) was significantly lower in the ASCs group at 36 hr (p = 0.003). Pulmonary shunt fraction tended to be lower in the ASCs group as compared to the control group. GFP-labelled ASCs were found on the surface of trachea epithelium 48 hrs after injury. The viability of ASCs in BALF was significantly lower than those exposed to the control vehicle solution. CONCLUSION: Nebulized ASCs moderately improved pulmonary function and delayed the onset of ARDS.

The physiologic responses to a fluid bolus administration in old and young healthy adults.

BACKGROUND: Organ function is known to decline with age. Optimizing cardiac, pulmonary and renal function in older adults has led to significant improvements in perioperative care. However, when substantial blood loss and fluid shifts occur, perioperative outcomes still remains poor, especially in older adults. We suspect that this could be due to age-related changes in endothelial function-an organ controlling the transport of fluid and solutes. The capillary filtration coefficient (CFC) is an important determinant of fluid transport. The CFC can be measured in vivo, which provides a tool to estimate endothelial barrier function. We have previously shown that the CFC increases when giving a fluid bolus resulting in increased vascular and extravascular volume expansion, in young adults. This study aimed to compare the physiologic determinants of fluid distribution in young versus older adults so that clinicians can best optimize perioperative fluid therapy. METHODS: Ten healthy young volunteers (ages 21-35) and nine healthy older volunteers (ages 60-75) received a 10 mL/kg fluid bolus over the course of twenty minutes. Hemodynamics, systolic and diastolic heart function, fluid volumetrics and microcirculatory determinants were measured before, during, and after the fluid bolus. RESULTS: Diastolic function was reduced in older versus younger adults before and after fluid bolus (P < 0.01). Basal CFC and plasma oncotic pressure were lower in the older versus younger adults. Further, CFC did not increase in older adults following the fluid bolus, whereas it did in younger adults (p < 0.05). Cumulative urinary output, while lower in older adults, was not significantly different (p = 0.059). Mean arterial pressure and systemic vascular resistance were elevated in the older versus younger adults (p < 0.05). CONCLUSION: Older adults show a less reactive CFC to a fluid bolus, which could reduce blood to tissue transport of fluid. Diastolic dysfunction likely contributes to fluid maldistribution in older adults.

Mechanistic insights gained from cell and molecular analysis of the neuroprotective potential of bioactive natural compounds in an immortalized hippocampal cell line.

Evaluating novel compounds for neuroprotective effects in animal models of traumatic brain injury (TBI) is a protracted, labor-intensive and costly effort. However, the present lack of effective treatment options for TBI, despite decades of research, shows the critical need for alternative methods for screening new drug candidates with neuroprotective properties. Because natural products have been a leading source of new therapeutic agents for human diseases, we used an in vitro model of stretch injury to rapidly assess pro-survival effects of three bioactive compounds, two isolated from natural products (clovanemagnolol [CM], vinaxanthone [VX]) and the third, a dietary compound (pterostilbene [PT]) found in blueberries. The stretch injury experiments were not used to validate drug efficacy in a comprehensive manner but used primarily, as proof-of-principle, to demonstrate that the neuroprotective potential of each bioactive agent can be quickly assessed in an immortalized hippocampal cell line in lieu of comprehensive testing in animal models of TBI. To gain mechanistic insights into potential molecular mechanisms of neuroprotective effects, we performed a pathway-specific PCR array analysis of the effects of CM on the rat hippocampus and microRNA sequencing analysis of the effects of VX and PT on cultured hippocampal progenitor neurons. We show that the neuroprotective properties of these natural compounds are associated with altered expression of several genes or microRNAs that have functional roles in neurodegeneration or cell survival. Our approach could help in quickly assessing multiple natural products for neuroprotective properties and expedite the process of new drug discovery for TBI therapeutics.