Inhibition of LRRK2-Rab10 Pathway Improves Secondary Brain Injury After Surgical Brain Injury in Rats

Leucine-rich repeat kinase 2 (LRRK2) is considered as a potential target for the treatment of Parkinson's disease. This protein is expressed in the brain and has been associated with various diseases and lysosomal maintenance. Rab10 is a member of the Rab protein GTPase family that has been recently shown to be a kinase substrate of LRRK2. In addition, LRRK2 and its kinase substrate Rab10 constitute a key stress response pathway during lysosomal overload stress. This study aimed to investigate the potential role and mechanism underlying LRRK2 and its kinase substrate Rab10 involving surgical brain injury (SBI). One hundred and forty-four male Sprague-Dawley rats were examined using an SBI model, and some had received the LRRK2-specific inhibitor PF-06447475. Thereafter, western blotting, immunofluorescence, brain water content analysis, neuronal apoptosis assay, and neurological score analysis were conducted. The results showed that after SBI, LRRK2 and phosphorylated Rab10 (p-Rab10) expression in neuronal cells were upregulated, and administration of PF-06447475 significantly reduced neuronal apoptosis, neuroinflammation, and brain water content 12 h after SBI and improved neurological deficit 72 h after SBI, which is related to the decreased expression of LRRK2 and p-Rab10, and the lessening of lysosomal overload stress. Our research suggests that the inhibition of LRRK2 can effectively interfere with the role of p-Rab10 in promoting the secretion of lysosomal hydrolase in lysosomal overload stress after SBI, thereby reducing neuronal apoptosis and inflammation after SBI and playing a major role in brain protection.

Brain water as a function of age and weight in normal rats

Rats are frequently used for studying water content of normal and injured brain, as well as changes in response to various osmotherapeutic regimens. Magnetic resonance imaging in humans has shown that brain water content declines with age as a result of progressive myelination and other processes. The purpose of this study was to quantify changes in brain water content during rat development and aging. Brain water content was measured by standard techniques in 129 normal male Sprague-Dawley rats that ranged in age (weight) from 13 to 149 days (18 to 759 g). Overall, the results demonstrated a decrease in water content from 85.59% to 76.56% with increasing age (weight). Nonlinear allometric functions relating brain water to age and weight were determined. These findings provide age-related context for prior rat studies of brain water, emphasize the importance of using similarly aged controls in studies of brain water, and indicate that age-related changes in brain water content are not specific to humans.

Sevoflurane Inhibits Traumatic Brain Injury-Induced Neuron Apoptosis via EZH2-Downregulated KLF4/p38 Axis

Traumatic brain injury (TBI) is characterized by physical damage to the brain tissues, ensuing transitory or permanent neurological dysfunction featured with neuronal loss and subsequent brain damage. Sevoflurane, a widely used halogenated anesthetic in clinical settings, has been reported to alleviate neuron apoptosis in TBI. Nevertheless, the underlying mechanism behind this alleviation remains unknown, and thus was the focus of the current study. First, Feeney models were established to induce TBI in rats. Subsequently, evaluation of the modified neurological severity scores, measurement of brain water content, Nissl staining, and TUNEL assay were employed to investigate the neuroprotective effects of sevoflurane. Immunofluorescence and Western blot analysis were further applied to detect the expression patterns of apoptosis-related proteins as well as the activation of the p38-mitogen-activated protein kinase (MAPK) signaling pathway within the lesioned cortex. Additionally, a stretch injury model comprising cultured neurons was established, followed by neuron-specific enolase staining and Sholl analysis. Mechanistic analyses were performed using dual-luciferase reporter gene and chromatin immunoprecipitation assays. The results demonstrated sevoflurane treatment brought about a decrease blood-brain barrier (BBB) permeability, brain water content, brain injury and neuron apoptosis, to improve neurological function. The neuroprotective action of sevoflurane could be attenuated by inactivation of the p38-MAPK signaling pathway. Mechanistically, sevoflurane exerted an inhibitory effect on neuron apoptosis by up-regulating enhancer of zeste homolog 2 (EZH2), which targeted Krüppel-like factor 4 (KLF4) and inhibited KLF4 transcription. Collectively, our findings indicate that sevoflurane suppresses neuron apoptosis induced by TBI through activation of the p38-MAPK signaling pathway via the EZH2/KLF4 axis, providing a novel mechanistic explanation for neuroprotection of sevoflurane in TBI.

A20 Establishes Negative Feedback With TRAF6/NF-κB and Attenuates Early Brain Injury After Experimental Subarachnoid Hemorrhage

Nuclear factor (NF)-κB–ty -50mediated neuroinflammation plays a crucial role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). As an important negative feedback regulator of NF-κB, A20 is essential for inflammatory homeostasis. Herein, we tested the hypothesis that A20 attenuates EBI by establishing NF-κB–associated negative feedback after experimental SAH. In vivo and in vitro models of SAH were established. TPCA-1 and lentivirus were used for NF-κB inhibition and A20 silencing/overexpression, respectively. Cellular localization of A20 in the brain was determined via immunofluorescence. Western blotting and enzyme-linked immunosorbent assays were applied to observe the expression of members of the A20/tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB pathway and inflammatory cytokines (IL-6, IL-1β, TNF-α). Evans blue staining, TUNEL staining, Nissl staining, brain water content, and modified Garcia score were performed to evaluate the neuroprotective effect of A20. A20 expression by astrocytes, microglia, and neurons was increased at 24 h after SAH. A20 and inflammatory cytokine levels were decreased while TRAF6 expression was elevated after NF-κB inhibition. TRAF6, NF-κB, and inflammatory cytokine levels were increased after A20 silencing but suppressed with A20 overexpression. Also, Bcl-2, Bax, MMP-9, ZO-1 protein levels; Evans blue, TUNEL, and Nissl staining; brain water content; and modified Garcia score showed that A20 exerted a neuroprotective effect after SAH. A20 expression was regulated by NF-κB. In turn, increased A20 expression inhibited TRAF6 and NF-κB to reduce the subsequent inflammatory response. Our data also suggest that negative feedback regulation mechanism of the A20/TRAF6/NF-κB pathway and the neuroprotective role of A20 to attenuate EBI after SAH.

Sex Differences in Cerebral Edema after Experimental Traumatic Brain Injury

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. Cerebral edema following TBI is known to play a critical role in injury severity and prognosis. In the current study we used multimodal magnetic resonance imaging (MRI) to assess cerebral edema 24 hours after unilateral contusive TBI in male and female rats. We then directly quantified brain water content in the same subjects ex vivo. We found that in male rats, the injured cortex had higher brain water content and lower apparent diffusion coefficient (ADC) values compared with the contralateral side. Females did not show hemispheric differences for these measures. However, both males and females had similarly elevated T2 values in the injured cortex compared with the contralateral side. A strong correlation was observed between brain water content and T2 values in the injured cortex in male rats, but not in females. These findings raise questions about the clinical interpretation of radiological findings pertinent to edema in female TBI patients. A more mechanistic understanding of sex differences and similarities in TBI pathophysiology will help improve patient management and the development of effective treatment strategies for TBI in men and women.

Mechanism of Baicalein in Brain Injury After Intracerebral Hemorrhage by Inhibiting the ROS/NLRP3 Inflammasome Pathway

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high disability/mortality. Baicalein has strong anti-inflammatory activity. This study aims to explore the mechanism of baicalein on brain injury after ICH. The model of brain injury after ICH was established by collagenase induction, followed by the evaluation of neurological severity, brain water content, the degenerated neurons, neuronal apoptosis and reactive oxygen species (ROS). The ICH model was treated with baicalein and silencing NLRP3 to detect brain injury. The expression of NLRP3 inflammasome was detected after treatment with ROS scavenger. The expression of oxidative stress markers and inflammatory factors were detected, and the levels of components in NLRP3 inflammasome were detected. Baicalein reduced the damage of nervous system, lesion surface, brain water content and apoptosis. Baicalein inhibited malondialdehyde and increased IL-10 by inhibiting ROS in brain tissue after ICH. Baicalein inhibited the high expression of NLRP3 inflammasome in ICH. ROS scavenger inhibited the NLRP3 inflammatory response by inhibiting ROS levels. Silencing NLRP3 alleviated the brain injury after ICH by inhibiting excessive oxidative stress and inflammatory factors. Overall, baicalein alleviated the brain injury after ICH by inhibiting ROS and NLRP3 inflammasome.

Glibenclamide does not improve outcome following severe collagenase-induced intracerebral hemorrhage in rats

Intracerebral hemorrhage (ICH) is a devastating insult with few effective treatments. Edema and raised intracranial pressure contribute to poor outcome after ICH. Glibenclamide blocks the sulfonylurea 1 transient receptor potential melastatin 4 (Sur1-Trpm4) channel implicated in edema formation. While glibenclamide has been found to improve outcome and reduce mortality in animal models of severe ischemic stroke, in ICH the effects are less clear. In our previous study, we found no benefit after a moderate-sized bleed, while others have reported benefit. Here we tested the hypothesis that glibenclamide may only be effective in severe ICH, where edema is an important contributor to outcome. Glibenclamide (10 μg/kg loading dose, 200 ng/h continuous infusion) was administered 2 hours post-ICH induced by collagenase injection into the striatum of adult rats. A survival period of 24 hours was maintained for experiments 1–3, and 72 hours for experiment 4. Glibenclamide did not affect hematoma volume (~81 μL) or other safety endpoints (e.g., glucose levels), suggesting the drug is safe. However, glibenclamide did not lessen striatal edema (~83% brain water content), ionic dyshomeostasis (Na+, K+), or functional impairment (e.g., neurological deficits (median = 10 out of 14), etc.) at 24 hours. It also did not affect edema at 72 h (~86% brain water content), or overall mortality rates (25% and 29.4% overall in vehicle vs. glibenclamide-treated severe strokes). Furthermore, glibenclamide appears to worsen cytotoxic edema in the peri-hematoma region (cell bodies were 46% larger at 24 h, p = 0.0017), but no effect on cell volume or density was noted elsewhere. Overall, these findings refute our hypothesis, as glibenclamide produced no favorable effects following severe ICH.

Metabolic Insight Into the Neuroprotective Effect of Tao-He-Cheng-Qi (THCQ) Decoction on ICH Rats Using Untargeted Metabolomics

Tao-He-Cheng-Qi decoction (THCQ) is an effective traditional Chinese medicine used to treat intracerebral hemorrhage (ICH). This study was performed to investigate the possible neuroprotective effect of THCQ decoction on secondary brain damage in rats with intracerebral hemorrhage and to elucidate the potential mechanism based on a metabolomics approach. Sprague-Dawley (SD) rats were randomly divided into five groups: the sham group, collagenase-induced ICH model group, THCQ low-dose (THCQ-L)-treated group, THCQ moderate-dose (THCQ-M)-treated group and THCQ high-dose (THCQ-H)-treated group. Following 3 days of treatment, behavioral changes and histopathological lesions in the brain were estimated. Untargeted metabolomics analysis with multivariate statistics was performed by using ultrahigh-performance liquid chromatography–mass spectrometry (UPLC-Q-Exactive Orbitrap MS). THCQ treatment at two dosages (5.64 and 11.27 g/kg·d) remarkably improved behavior (p < 0.05), brain water content (BMC) and hemorheology (p < 0.05) and improved brain nerve tissue pathology and inflammatory infiltration in ICH rats. Moreover, a metabolomic analysis demonstrated that the serum metabolic profiles of ICH patients were significantly different between the sham group and the ICH-induced model group. Twenty-seven biomarkers were identified that potentially predict the clinical benefits of THCQ decoction. Of these, 4 biomarkers were found to be THCQ-H group-specific, while others were shared between two clusters. These metabolites are mainly involved in amino acid metabolism and glutamate-mediated cell excitotoxicity, lipid metabolism-mediated oxidative stress, and mitochondrial dysfunction caused by energy metabolism disorders. In addition, a correlation analysis showed that the behavioral scores, brain water content and hemorheology were correlated with levels of serum metabolites derived from amino acid and lipid metabolism. In conclusion, the results indicate that THCQ decoction significantly attenuates ICH-induced secondary brain injury, which could be mediated by improving metabolic disorders in cerebral hemorrhage rats.

The Antiedematous Effect of Exogenous Lactate Therapy in Traumatic Brain Injury: A Physiological and Mechanistic Approach

Background Sodium lactate (SL) has been described as an efficient therapy in treating raised intracranial pressure (ICP). However, the precise mechanism by which SL reduces intracranial hypertension is not well defined. An antiedematous effect has been proposed but never demonstrated. In this context, the involvement of chloride channels, aquaporins, or K–Cl cotransporters has also been suggested, but these mechanisms have never been assessed when using SL. Methods In a rat model of traumatic brain injury (TBI), we compared the effect of SL versus mannitol 20% on ICP, cerebral tissue oxygen pressure, and brain water content. We attempted to clarify the involvement of chloride channels in the antiedematous effects associated with lactate therapy in TBI. Results An equimolar single bolus of SL and mannitol significantly reduced brain water content and ICP and improved cerebral tissue oxygen pressure 4 h after severe TBI. The effect of SL on brain water content was much longer than that of mannitol and persisted at 24 h post TBI. Western blot and immunofluorescence staining analyses performed 24 h after TBI revealed that SL infusion is associated with an upregulation of aquaporin 4 and K–Cl cotransporter 2. Conclusions SL is an effective therapy for treating brain edema after TBI. This study suggests, for the first time, the potential role of chloride channels in the antiedematous effect induced by exogenous SL.

Brain Water as a Function of Age and Weight in Normal Rats

Rats are frequently used to study water content of normal and injured brain, as well as changes in response to various osmotherapeutic regimens. Magnetic resonance imaging in humans has shown that brain water content declines with age as a result of progressive myelination. The purpose of this study was to quantify changes in brain water content during rat development and aging. Brain water content was measured by standard techniques in 129 normal male Sprague-Dawley rats that ranged in age (weight) from 13 to 149 days (18 to 759 g). Overall, the results demonstrated a decrease from 85.59% to 76.56% water content with increasing age (weight). Nonlinear allometric functions relating brain water to age and weight were determined. These findings provide age-related context for prior rat studies of brain water, emphasize the importance of using similarly aged controls in studies of brain water, and indicate that age-related changes in brain water content are not specific to humans.