Intranasal delivery of mitochondria targeted neuroprotective compounds for traumatic brain injury: screening based on pharmacological and physiological properties.

Targeting drugs to the mitochondrial level shows great promise for acute and chronic treatment of traumatic brain injury (TBI) in both military and civilian sectors. Perhaps the greatest obstacle to the successful delivery of drug therapies is the blood brain barrier (BBB). Intracerebroventricular and intraparenchymal routes may provide effective delivery of small and large molecule therapies for preclinical neuroprotection studies. However, clinically these delivery methods are invasive, and risk inadequate exposure to injured brain regions due to the rapid turnover of cerebral spinal fluid. The direct intranasal drug delivery approach to therapeutics holds great promise for the treatment of central nervous system (CNS) disorders, as this route is non-invasive, bypasses the BBB, enhances the bioavailability, facilitates drug dose reduction, and reduces adverse systemic effects. Using the intranasal method in animal models, researchers have successfully reduced stroke damage, reversed Alzheimer's neurodegeneration, reduced anxiety, improved memory, and delivered neurotrophic factors and neural stem cells to the brain. Based on literature spanning the past several decades, this review aims to highlight the advantages of intranasal administration over conventional routes for TBI, and other CNS disorders. More specifically, we have identified and compiled a list of most relevant mitochondria-targeted neuroprotective compounds for intranasal administration based on their mechanisms of action and pharmacological properties. Further, this review also discusses key considerations when selecting and testing future mitochondria-targeted drugs given intranasally for TBI.

PEG hydrogel containing dexamethasone-conjugated hyaluronic acid reduces secondary injury and improves motor function in a rat moderate TBI model.

Traumatic brain injury (TBI) leads to long-term impairments in motor and cognitive function. TBI initiates a secondary injury cascade including a neuro-inflammatory response that is detrimental to tissue repair and limits recovery. Anti-inflammatory corticosteroids such as dexamethasone can reduce the deleterious effects of secondary injury; but challenges associated with dosing, administration route, and side effects have hindered their clinical application. Previously, we developed a hydrolytically degradable hydrogel (PEG-bis-AA/HA-DXM) composed of poly (ethylene) glycol-bis-(acryloyloxy acetate) (PEG-bis-AA) and dexamethasone-conjugated hyaluronic acid (HA-DXM) for local and sustained dexamethasone delivery. In this study, we evaluated the effect of locally applied PEG-bis-AA/HA-DXM hydrogel on secondary injury and motor function recovery after moderate controlled cortical impact (CCI) TBI. Hydrogel treatment significantly improved motor function evaluated by beam walk and rotarod tests compared to untreated rats over 7 days post-injury (DPI). We also observed that the hydrogel treatment reduced lesion volume, inflammatory response, astrogliosis, apoptosis, and increased neuronal survival compared to untreated rats at 7 DPI. These results suggest that PEG-bis-AA/HA-DXM hydrogels can mitigate secondary injury and promote motor functional recovery following moderate TBI.

EFFECTS OF TRANEXAMIC ACID ON NEUROPATHOLOGY, ELECTROENCEPHALOGRAPHY, AND CEREBRAL FIBRIN DEPOSITION IN A RAT MODEL OF POLYTRAUMA WITH CONCOMITANT PENETRATING TRAUMATIC BRAIN INJURY.

ABSTRACT: Several studies have demonstrated the clinical utility of tranexamic acid (TXA) for use in trauma patients presenting with significant hemorrhage. Tranexamic acid is an antifibrinolytic that inhibits plasminogen activation, and plasmin activity has been shown to mitigate blood loss and reduce all-cause mortality in the absence of adverse vascular occlusive events. Recent clinical developments indicate TXA is safe to use in patients with concomitant traumatic brain injury (TBI); however, the prehospital effects are not well understood. Importantly, TXA has been associated with seizure activity. Therefore, this study sought to evaluate the effects of early administration of TXA on neurological recovery and electroencephalogram (EEG) abnormalities following penetrating TBI with concomitant hypoxemia and hemorrhagic shock. We hypothesized that early administration of TXA will provide hemodynamic stabilization and reduce intracerebral hemorrhage, which will result in improved neurological function. To test this hypothesis, Sprague-Dawley rats received a unilateral, frontal penetrating ballistic-like brain injury by inserting a probe into the frontal cortex of the anesthetized rat. Five minutes following brain injury, animals underwent 30 min of respiratory distress and 30 min of hemorrhage. Upon completion of the hemorrhage phase, animals received the initial dose of drug intravenously over 10 min after which the prehospital phase was initiated. During the prehospital phase, animals received autologous shed whole blood as needed to maintain a MAP of 65 mm Hg. After 90 min, "in-hospital" resuscitation was performed by administering the remaining shed whole blood providing 100% oxygen for 15 min. Upon recovery from surgery, animals were administered their second dose of vehicle or TXA intravenously over 8 h. Tranexamic acid induced an early improvement in neurologic deficit, which was statistically significant compared with vehicle at 24, 48, and 72 h at three doses tested. Analysis of cerebral hemoglobin content and intracerebral lesion progression revealed 100 mg/kg provided the optimal effects for improvement of neuropathology and was continued for determination of adverse treatment effects. We observed no exacerbation of cerebral thrombosis, but TXA treatment caused an increased risk of EEG abnormalities. These results suggest that TXA following polytrauma with concomitant brain injury may provide mild neuroprotective effects by preventing lesion progression, but this may be associated with an increased risk of abnormal EEG patterns. This risk may be associated with TXA inhibition of glycine receptors and may warrant additional considerations during the use of TXA in patients with severe TBI.

Choice of Whole Blood versus Lactated Ringer's Resuscitation Modifies the Relationship between Blood Pressure Target and Functional Outcome after Traumatic Brain Injury plus Hemorrhagic Shock in Mice.

Civilian traumatic brain injury (TBI) guidelines recommend resuscitation of patients with hypotensive TBI with crystalloids. Increasing evidence, however, suggests that whole blood (WB) resuscitation may improve physiological and survival outcomes at lower resuscitation volumes, and potentially at a lower mean arterial blood pressure (MAP), than crystalloid after TBI and hemorrhagic shock (HS). The objective of this study was to assess whether WB resuscitation with two different MAP targets improved behavioral and histological outcomes compared with lactated Ringer's (LR) in a mouse model of TBI+HS. Anesthetized mice (n = 40) underwent controlled cortical impact (CCI) followed by HS (MAP = 25-27 mm Hg; 25 min) and were randomized to five groups for a 90 min resuscitation: LR with MAP target of 70 mm Hg (LR70), LR60, WB70, WB60, and monitored sham. Mice received a 20 mL/kg bolus of LR or autologous WB followed by LR boluses (10 mL/kg) every 5 min for MAP below target. Shed blood was reinfused after 90 min. Morris Water Maze testing was performed on days 14-20 post-injury. Mice were euthanized (21 d) to assess contusion and total brain volumes. Latency to find the hidden platform was greater versus sham for LR60 (p < 0.002) and WB70 (p < 0.007) but not LR70 or WB60. The WB resuscitation did not reduce contusion volume or brain tissue loss. The WB targeting a MAP of 60 mm Hg did not compromise function versus a 70 mm Hg target after CCI+HS, but further reduced fluid requirements (p < 0.03). Using LR, higher achieved MAP was associated with better behavioral performance (rho = -0.67, p = 0.028). Use of WB may allow lower MAP targets without compromising functional outcome, which could facilitate pre-hospital TBI resuscitation.

Prehospital Whole Blood Resuscitation Reduces Fluid Requirement While Maintaining Critical Physiology in a Model of Penetrating Traumatic Brain Injury and Hemorrhage: Implications on Resource-Limited Combat Casualty Care.

ABSTRACT: Prehospital resuscitation using whole blood (WB) is the standard of care for hemorrhagic shock (HS) but there is no consensus recommendation for resuscitation in the presence of traumatic brain injury (TBI) due to a lack of sufficient evidence. In order to evaluate the optimal resuscitation strategies for TBI+HS, Sprague-Dawley rats were randomized into four groups based on resuscitation fluid and prehospital mean arterial pressure (MAP) threshold (n = 9-10/group): Lactated Ringer's (LR)-60 mm Hg (LR60), LR-70 mm Hg (LR70), WB-60 mm Hg (WB60), WB-70 mm Hg (WB70). All groups received a frontal penetrating ballistic-like brain injury followed by a 35-min period of HS. During the prehospital phase, rats received an initial bolus of resuscitation fluid (WB or LR) followed by LR as needed to maintain MAP above the designated threshold for 90 min. During the in-hospital phase, rats received definitive resuscitation with shed WB. Physiological parameters were recorded continuously and cerebral edema was measured at 3 and 24 h postinjury. The WB60 group demonstrated a significantly lower prehospital fluid requirement compared WB70, LR60, and LR70 (P < 0.05). Compared to the respective LR groups, both the WB60 and WB70 groups also demonstrated improved MAP, cerebral perfusion pressure, brain tissue oxygen tension, and cerebral edema. The edema benefits were observed at 3 h, but not 24 h postinjury, and were localized to the injury site. Together, these results provide evidence that prehospital WB resuscitation and lower MAP resuscitation thresholds can reduce the prehospital fluid requirement while still maintaining critical cerebral physiology in a model of HS and concomitant TBI.

Multifaceted Benefit of Whole Blood Versus Lactated Ringer's Resuscitation After Traumatic Brain Injury and Hemorrhagic Shock in Mice.

BACKGROUND: Despite increasing use in hemorrhagic shock (HS), whole blood (WB) resuscitation for polytrauma with traumatic brain injury (TBI) is largely unexplored. Current TBI guidelines recommend crystalloid for prehospital resuscitation. Although WB outperforms lactated Ringer's (LR) in increasing mean arterial pressure (MAP) in TBI + HS models, effects on brain tissue oxygenation (PbtO2), and optimal MAP remain undefined. METHODS: C57BL/6 mice (n = 72) underwent controlled cortical impact followed by HS (MAP = 25-27 mmHg). Ipsilateral hippocampal PbtO2 (n = 40) was measured by microelectrode. Mice were assigned to four groups (n = 18/group) for "prehospital" resuscitation (90 min) with LR or autologous WB, and target MAPs of 60 or 70 mmHg (LR60, WB60, LR70, WB70). Additional LR (10 ml/kg) was bolused every 5 min for MAP below target. RESULTS: LR requirements in WB60 (7.2 ± 5.0 mL/kg) and WB70 (28.3 ± 9.6 mL/kg) were markedly lower than in LR60 (132.8 ± 5.8 mL/kg) or LR70 (152.2 ± 4.8 mL/kg; all p < 0.001). WB70 MAP (72.5 ± 2.9 mmHg) was higher than LR70 (59.8 ± 4.0 mmHg, p < 0.001). WB60 MAP (68.7 ± 4.6 mmHg) was higher than LR60 (53.5 ± 3.2 mmHg, p < 0.001). PbtO2 was higher in WB60 (43.8 ± 11.6 mmHg) vs either LR60 (25.9 ± 13.0 mmHg, p = 0.04) or LR70 (24.1 ± 8.1 mmHg, p = 0.001). PbtO2 in WB70 (40.7 ± 8.8 mmHg) was higher than in LR70 (p = 0.007). Despite higher MAP in WB70 vs WB60 (p = .002), PbtO2 was similar. CONCLUSION: WB resuscitation after TBI + HS results in robust improvements in brain oxygenation while minimizing fluid volume when compared to standard LR resuscitation. WB resuscitation may allow for a lower prehospital MAP without compromising brain oxygenation when compared to LR resuscitation. Further studies evaluating the effects of these physiologic benefits on outcome after TBI with HS are warranted, to eventually inform clinical trials.

Cerium Oxide Nanoparticles Improve Outcome after In Vitro and In Vivo Mild Traumatic Brain Injury.

Mild traumatic brain injury results in aberrant free radical generation, which is associated with oxidative stress, secondary injury signaling cascades, mitochondrial dysfunction, and poor functional outcome. Pharmacological targeting of free radicals with antioxidants has been examined as an approach to treatment, but has met with limited success in clinical trials. Conventional antioxidants that are currently available scavenge a single free radical before they are destroyed in the process. Here, we report for the first time that a novel regenerative cerium oxide nanoparticle antioxidant reduces neuronal death and calcium dysregulation after in vitro trauma. Further, using an in vivo model of mild lateral fluid percussion brain injury in the rat, we report that cerium oxide nanoparticles also preserve endogenous antioxidant systems, decrease macromolecular free radical damage, and improve cognitive function. Taken together, our results demonstrate that cerium oxide nanoparticles are a novel nanopharmaceutical with potential for mitigating neuropathological effects of mild traumatic brain injury and modifying the course of recovery.

The Effects of Balloon Occlusion of the Aorta on Cerebral Blood Flow, Intracranial Pressure, and Brain Tissue Oxygen Tension in a Rodent Model of Penetrating Ballistic-Like Brain Injury.

Trauma is among the leading causes of death in the United States. Technological advancements have led to the development of resuscitative endovascular balloon occlusion of the aorta (REBOA) which offers a pre-hospital option to non-compressible hemorrhage control. Due to the prevalence of concomitant traumatic brain injury (TBI), an understanding of the effects of REBOA on cerebral physiology is critical. To further this understanding, we employed a rat model of penetrating ballistic-like brain injury (PBBI). PBBI produced an injury pattern within the right frontal cortex and striatum that replicates the pathology from a penetrating ballistic round. Aortic occlusion was initiated 30 min post-PBBI and maintained continuously (cAO) or intermittently (iAO) for 30 min. Continuous measurements of mean arterial pressure (MAP), intracranial pressure (ICP), cerebral blood flow (CBF), and brain tissue oxygen tension (PbtO2) were recorded during, and for 60 min following occlusion. PBBI increased ICP and decreased CBF and PbtO2. The arterial balloon catheter effectively occluded the descending aorta which augmented MAP in the carotid artery. Despite this, CBF levels were not changed by aortic occlusion. iAO caused sustained adverse effects to ICP and PbtO2 while cAO demonstrated no adverse effects on either. Temporary increases in PbtO2 were observed during occlusion, along with restoration of sham levels of ICP for the remainder of the recordings. These results suggest that iAO may lead to prolonged cerebral hypertension following PBBI. Following cAO, ICP, and PbtO2 levels were temporarily improved. This information warrants further investigation using TBI-polytrauma model and provides foundational knowledge surrounding the non-hemorrhage applications of REBOA including neurogenic shock and stroke.

Cellular Mechanisms and Behavioral Outcomes in Blast-Induced Neurotrauma: Comparing Experimental Setups.

Blast-induced neurotrauma (BINT) has increased in incidence over the past decades and can result in cognitive issues that have debilitating consequences. The exact primary and secondary mechanisms of injury have not been elucidated and appearance of cellular injury can vary based on many factors, such as blast overpressure magnitude and duration. Many methodologies to study blast neurotrauma have been employed, ranging from open-field explosives to experimental shock tubes for producing free-field blast waves. While there are benefits to the various methods, certain specifications need to be accounted for in order to properly examine BINT. Primary cell injury mechanisms, occurring as a direct result of the blast wave, have been identified in several studies and include cerebral vascular damage, blood-brain barrier disruption, axonal injury, and cytoskeletal damage. Secondary cell injury mechanisms, triggered subsequent to the initial insult, result in the activation of several molecular cascades and can include, but are not limited to, neuroinflammation and oxidative stress. The collective result of these secondary injuries can lead to functional deficits. Behavioral measures examining motor function, anxiety traits, and cognition/memory problems have been utilized to determine the level of injury severity. While cellular injury mechanisms have been identified following blast exposure, the various experimental models present both concurrent and conflicting results. Furthermore, the temporal response and progression of pathology after blast exposure have yet to be detailed and remain unclear due to limited resemblance of methodologies. This chapter summarizes the current state of blast neuropathology and emphasizes the need for a standardized preclinical model of blast neurotrauma.

Astrocyte Reactivity Following Blast Exposure Involves Aberrant Histone Acetylation.

Blast induced neurotrauma (BINT) is a prevalent injury within military and civilian populations. The injury is characterized by persistent inflammation at the cellular level which manifests as a multitude of cognitive and functional impairments. Epigenetic regulation of transcription offers an important control mechanism for gene expression and cellular function which may underlie chronic inflammation and result in neurodegeneration. We hypothesize that altered histone acetylation patterns may be involved in blast induced inflammation and the chronic activation of glial cells. This study aimed to elucidate changes to histone acetylation occurring following injury and the roles these changes may have within the pathology. Sprague Dawley rats were subjected to either a 10 or 17 psi blast overpressure within an Advanced Blast Simulator (ABS). Sham animals underwent the same procedures without blast exposure. Memory impairments were measured using the Novel Object Recognition (NOR) test at 2 and 7 days post-injury. Tissues were collected at 7 days for Western blot and immunohistochemistry (IHC) analysis. Sham animals showed intact memory at each time point. The novel object discrimination decreased significantly between two and 7 days for each injury group (p < 0.05). This is indicative of the onset of memory impairment. Western blot analysis showed glial fibrillary acidic protein (GFAP), a known marker of activated astrocytes, was elevated in the prefrontal cortex (PFC) following blast exposure for both injury groups. Analysis of histone protein extract showed no changes in the level of any total histone proteins within the PFC. However, acetylation levels of histone H2b, H3, and H4 were decreased in both groups (p < 0.05). Co-localization immunofluorescence was used to further investigate any potential correlation between decreased histone acetylation and astrocyte activation. These experiments showed a similar decrease in H3 acetylation in astrocytes exposed to a 17 psi blast but not a 10 psi blast. Further investigation of gene expression by polymerase chain reaction (PCR) array, showed dysregulation of several cytokine and cytokine receptors that are involved in neuroinflammatory processes. We have shown aberrant histone acetylation patterns involved in blast induced astrogliosis and cognitive impairments. Further understanding of their role in the injury progression may lead to novel therapeutic targets.

Blast induced neurotrauma causes overpressure dependent changes to the DNA methylation equilibrium.

Traumatic brain injury (TBI) has a high prevalence in our society and often leads to morbidity and mortality. TBI also occurs frequently in a military setting where exposure to blast waves is common. Abnormal gene expression involved with oxidative stress, inflammation and neuronal apoptosis has been well documented following blast induced neurotrauma (BINT). Altered epigenetic transcriptional regulation through DNA methylation has been implicated in the pathology of the injury. Imbalance between DNA methylation and DNA demethylation may lead to altered methylation patterns and subsequent changes in gene transcription. DNA methyltransferase enzymes (DNMT1, DNMT3a, and DNMT3b) are responsible for the addition of methyl groups to DNA, DNA methylation. Whereas the combined function of ten-eleven translocation enzymes (TET1, TET2, and TET3) and thymine-DNA glycosylase (TDG) result in the removal of methyl groups from DNA, DNA demethylation. We used an established rodent model of BINT to assess changes in DNA methylation and demethylation enzymes following injury. Three different blast overpressures were investigated (10, 17 and 23psi). Gene expression was investigated in the prefrontal cortex and hippocampus two weeks following injury. We observed DNMT, TET and TDG expression changes between pressure groups and brain regions. The hippocampus was more vulnerable to enzyme expression changes than the prefrontal cortex, which correlated with aberrant DNA methylation. A significant negative correlation was found between global DNA methylation and the magnitude of blast overpressure exposure. Through transcriptional regulation, altered DNA methylation patterns may offer insight into the characteristic outcomes associated with the injury pathology including inflammation, oxidative stress and apoptosis. As such, these enzymes may be important targets to future therapeutic intervention strategies.

Blast Induced Neurotrauma Leads To Changes In The Epigenome.

Blast induced neurotrauma (BINT) leads to widespread aberrant gene expression and molecular changes resulting in cognitive impairment. Enzymes such as HDAC2, HDAC6, SIRT1, DNMT1, DNMT3a and DNMT3b control histone acetylation and DNA methylation which play a major role in regulation of the transcriptome. Changes in the expression of these enzymes have been implicated in the pathology of traumatic brain injury (TBI) and Alzheimer’s disease (AD). We hypothesize that blast exposure will lead to changes in the expression of these enzymes which play a key role in injury progression and pathology. This study looked to identify epigenome changes in the acute stages of BINT using an established rodent model. Real time polymerase chain reaction and Western blot analyses were used to assess gene expression and protein level changes compared to sham. No significant changes were seen 24 hours after blast exposure. However, several changes were observed at 72 hours following blast exposure. There was a significant increase in expression of HDAC2 and HDAC6 in the hippocampus which correlated with elevated HDAC2 protein levels. SIRT1, DNMT3a and DNMT3b levels were all reduced in the hippocampus. In the medial prefrontal cortex, DNMT1 and DNMT3b were significantly reduced. The results indicated that blast exposure causes acute changes in gene expression and protein levels of epigenetic markers which correlate with changes observed in AD pathology. These epigenomic changes could provide novel targets for therapeutic interventions following BINT.