Wet-dry Weight Method Research Articles

Long-term hypothermia amplified neuroprotection by antagonizing intracranial pressure rebound after severe traumatic brain injury in rats.

Long-term hypothermia has been reported to prevent intracranial pressure (ICP) rebound in clinical patients, but the duration for hypothermia and the corresponding ICP data are not available. This study investigated the optimal duration of long-term hypothermia in traumatic brain injury (TBI) rats, and observed the effect on ICP and neurological function. In this study, we established a rat severe TBI model with electronic Controlled Cortical Injury device, and implemented hypothermia (33 °C) for different durations. The motor function of the rats in each group was evaluated by beam walking test and inclined-grid climbing test, brain water content was calculated by the wet-dry weight method, Evan's blue staining was used to measure the blood-brain barrier (BBB) permeability, the change of hippocampal neurons was observed by Nissl staining, the expressions of BrdU, NeuN, and CD86 positive cells were detected by immunofluorescence staining, and the expressions of Bcl-2, Bax, iNOS, IL-10, and Arg-1 were detected by Western blot. We found that therapeutic hypothermia improved neurological recovery after TBI with declining ICP, reducing brain edema, decreasing BBB permeability, promoting neurogenesis, inhibiting apoptosis, and regulating inflammation. Moreover, 48 h hypothermia amplified the neuroprotective effect after injury on the basis of 4 or 24 h hypothermic treatment. Both 4 and 24 h hypothermia led to ICP rebound during or after rewarming, whereas 48 h hypothermia completely abolished ICP rebound. Our study suggests that long-term hypothermia amplifies neuroprotection after TBI by antagonizing ICP rebound.

MicroRNA-7 attenuates secondary brain injury following experimental intracerebral hemorrhage via inhibition of NLRP3

Background and purposeThe pathophysiological mechanisms underlying brain injury resulting from intracerebral hemorrhage (ICH) remain incompletely elucidated, and efficacious therapeutic interventions to enhance the prognosis of ICH patients are currently lacking. Previous research indicates that MicroRNA-7 (miR-7) can suppress the expression of Nod-like receptor protein 3 (NLRP3), thereby modulating neuroinflammation in Parkinson's disease pathogenesis. However, the potential regulatory effects miR-7 on NLRP3 inflammasome after ICH are yet to be established. This study aims to ascertain whether miR-7 mitigates secondary brain injury following experimental ICH by inhibiting NLRP3 and to investigate the underlying mechanisms. MethodsAn ICH model was established by stereotaxically injecting 100 μL of autologous blood into the right basal ganglia of Sprague-Dawley (SD) rats. Subsequently, these rats were allocated into three groups: sham, ICH + Vehicle, and ICH + miR-7, each comprising 18 animals. Twelve hours post-modeling, rats received intraventricular injections of 10 μL physiological saline, 10 μL phosphate, and 10 μL phosphate-buffered saline solution containing 0.5 nmol of miR-7 mimics, respectively. Neurological function was assessed on day three post-modeling, followed by euthanasia for brain tissue collection. Brain water content was determined using the dry-wet weight method. The expression of inflammatory cytokines in cerebral tissues surrounding the hematoma was analyzed through immunohistochemistry and Western blot assays. These cytokines were re-evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, bioinformatics tools were employed to predict miR-7′s binding to NLRP3. A wild-type luciferase reporter gene vector and a corresponding mutant vector were constructed, followed by transfection of miR-7 mimics into HEK293T cells to assess luciferase activity. ResultsOur study demonstrates that the administration of miR-7 mimics markedly reduced neurological function scores and attenuated brain edema in rats following ICH. A significant upregulation of NLRP3 expression in microglia/macrophage adjacent to the hematoma was observed, substantially reduced after the treatment with miR-7 mimics. Furthermore, this intervention ameliorated neurodegenerative changes and effectively decreased the protein and mRNA levels of pro-inflammatory cytokines, namely TNF-α, IL-1β, IL-6, and Caspase1, in the cerebral tissues proximate to the hematomas. In addition, miR-7 mimics distinctly inhibited the luciferase activity associated with the wild-type reporter gene, an effect not mirrored in its mutant variant. ConclusionsThe miR-7 suppressed NLRP3 expression in microglia/macrophage to reduce the production of inflammatory cytokines, leading to conducting certain neuroprotection post-ICH in rats.

Progesterone induces neuroprotection associated with immune/inflammatory modulation in experimental traumatic brain injury.

An imbalance of immune/inflammatory reactions aggravates secondary brain injury after traumatic brain injury (TBI) and can deteriorate clinical prognosis. So far, not enough therapeutic avenues have been found to prevent such an imbalance in the clinical setting. Progesterone has been shown to regulate immune/inflammatory reactions in many diseases and conveys a potential protective role in TBI. This study was designed to investigate the neuroprotective effects of progesterone associated with immune/inflammatory modulation in experimental TBI. A TBI model in adult male C57BL/6J mice was created using a controlled contusion instrument. After injury, the mice received consecutive progesterone therapy (8 mg/kg per day, i.p.) until euthanized. Neurological deficits were assessed via Morris water maze test. Brain edema was measured via the dry-wet weight method. Immunohistochemical staining and flow cytometry were used to examine the numbers of immune/inflammatory cells, including IBA-1 + microglia, myeloperoxidase + neutrophils, and regulatory T cells (Tregs). ELISA was used to detect the concentrations of IL-1β, TNF-α, IL-10, and TGF-β. Our data showed that progesterone therapy significantly improved neurological deficits and brain edema in experimental TBI, remarkably increased regulatory T cell numbers in the spleen, and dramatically reduced the activation and infiltration of inflammatory cells (microglia and neutrophils) in injured brain tissue. In addition, progesterone therapy decreased the expression of the pro-inflammatory cytokines IL-1β and TNF-α but increased the expression of the anti-inflammatory cytokine IL-10 after TBI. These findings suggest that progesterone administration could be used to regulate immune/inflammatory reactions and improve outcomes in TBI.

Inhibition of S100A9 alleviates neurogenic pulmonary edema after subarachnoid hemorrhage

Background and PurposeNeurogenic pulmonary edema (NPE) frequently arises as a complication subsequent to subarachnoid hemorrhage (SAH). Heterodimers of S100A8 and S100A9 are commonly formed, thereby initiating an inflammatory reaction through receptor binding on the cell surface. Paquinimod serves as a specific inhibitor of S100A9. The objective of this investigation is to assess the impact of Paquinimod administration and S100A9 knockout on NPE following SAH. MethodsIn this study, SAH models of C57BL/6J wild-type (WT) and S100A9 knockout mice were established through intravascular perforation. These models were then divided into several groups, including the WT-sham group, S100A9-KO-sham group, WT-SAH group, WT-SAH + Paquinimod group, and S100A9-KO-SAH group. After 24 h of SAH induction, pulmonary edema was assessed using the lung wet-dry weight method and Hematoxylin and eosin (HE) staining. Additionally, the expression levels of various proteins, such as interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), occludin, claudin-3, Bax, Bcl-2, TLR4, MYD88, and pNF-κB, in lung tissue were analyzed using western blot and immunofluorescence staining. Lung tissue apoptosis was detected by TUNEL staining. ResultsFirstly, our findings indicate that the knockout of S100A9 has a protective effect on early brain injury following subarachnoid hemorrhage (SAH). Additionally, the reduction of brain injury after SAH can also alleviate neurogenic pulmonary edema (NPE). Immunofluorescence staining and western blot analysis revealed that compared to SAH mice with wild-type S100A9 expression (WT-SAH), the lungs of S100A9 knockout SAH mice (S100A9-KO-SAH) and mice treated with Paquinimod exhibited decreased levels of inflammatory molecules (IL-1β and TNF-α) and increased levels of tight junction proteins. Furthermore, the knockout of S100A9 resulted in upregulated expression of the apoptotic-associated protein Bax and down-regulated expression of Bcl-2. Furthermore, a decrease in TLR4, MYD88, and phosphorylated pNF-κB was noted in S100A9-KO-SAH and Paquinimod treated mice, indicating the potential involvement of the TLR4/MYD88/NF-κB signaling pathway in the inhibition of the protective effect of S100A9 on NPE following SAH. ConclusionThe knockout of S100A9 not only ameliorated initial cerebral injury following subarachnoid hemorrhage (SAH), but also mitigated SAH-associated neurogenic pulmonary edema (NPE). Additionally, Paquinimod was found to diminish NPE. These findings imply a correlation between the central nervous system and peripheral organs, highlighting the potential of safeguarding the brain to mitigate harm to peripheral organs.

Low-dose LPS alleviates early brain injury after SAH by modulating microglial M1/M2 polarization via USP19/FOXO1/IL-10/IL-10R1 signaling

BackgroundLow-dose lipopolysaccharide (LPS) protects against early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the mechanism underlying the neuroprotective roles of low-dose LPS remain largely undefined. MethodsA SAH mice model was established and the pathological changes of brain were evaluated by wet-dry weight method, HE and Nissl staining, and blood-brain barrier (BBB) permeability assay. Cell apoptosis and inflammation were monitored by TUNEL, flow cytometry and ELISA assays. qRT-PCR, immunofluorescence and Western blot were used to detect the expression of microglial polarization-related or oxidative stress-associated markers. Bioinformatics analysis, luciferase and ChIP assays were employed to detect the direct association between FOXO1 and IL-10 promoter. The ubiquitination of FOXO1 in the in vitro SAH model was detected by co-IP. ResultsLow-dose LPS alleviated SAH-induced neurological dysfunction, brain edema, BBB disruption, damage in the hippocampus, neuronal apoptosis and inflammation via modulating microglial M1/M2 polarization by IL-10/IL-10R1 signaling. Mechanistic studies showed that FOXO1 acted as a transcriptional activator of IL-10. USP19 mediated the deubiquitination of FOXO1 to activate IL-10/IL-10R1 signaling, thereby regulating microglial M1/M2 polarization. Functional experiments revealed that low-dose LPS upregulated USP19 to modulate microglial M1/M2 polarization via FOXO1/IL-10/IL-10R1 signaling in SAH mice. ConclusionLow-dose LPS protected against EBI after SAH by modulating microglial M1/M2 polarization via USP19/FOXO1/IL-10/IL-10R1 signaling.

The Effect of Electrospinning Parameters on Morphological and Mechanical Properties of PAN-based Nanofibers Membrane

The electrospun nanofibers membranes (ENMs) have gained great attention due to their superior performance. However, the low mechanical strength of ENMs, such as the rigidity and low strength, limits their applications in many aspects which need adequate strength, such as water filtration. This work investigates the impact of electrospinning parameters on the properties of ENMs fabricated from polyacrylonitrile (PAN) solved in N, N-Dimethylformamide (DMF). The studied electrospinning parameters were polymer concentration, solution flow rate, collector rotating speed, and the distance between the needle and collector. The fabricated ENMs were characterized using scanning electron microscopy (SEM) to understand the surface morphology and estimate the average fiber sizes. The membrane porosity percentage was measured using the dry-wet weight method. Also, a dynamic mechanical analyzer was used to determine the mechanical strength properties (tensile strength and Young's modulus) (DMA). The obtained results revealed that the polymer concentration and flow rate mainly affect the porosity and fiber size in ENMs. Increasing the polymer concentration improves the strength and flexibility, while the flow rate did not show a clear effect on the mechanical strength of ENMs. Both fibers collecting speed and spinning distance did not clearly impact the membrane morphology. ENMs flexibility significantly increased with increasing the collector speed and decreasing the spinning distance. Strong and flexible ENMs with small fibers can be fabricated using 10% PAN/DMF at a flow rate of 1 mL/h, collector speed of 140 rpm, and spinning distance of 13 cm.

Dexmedetomidine (Dex) exerts protective effects on rat neuronal cells injured by cerebral ischemia/reperfusion via regulating the Sphk1/S1P signaling pathway

AimTo investigate the influence of dexmedetomidine (Dex) on cerebral ischemia/reperfusion (I/R)-injured rat neuronal cells by regulating the Sphk1/S1P pathway. MethodsThe rats were divided into the following groups, with 18 rats in each group categorized on the basis of random number tables: sham (Sham), I/R (I/R), Dex, Sphk1 inhibitor (PF-543), and Dex together with the Sphk1 agonist phorbol-12-myristate-13-acetate (Dex+PMA). The neurological functions of the rats were assessed by the Longa scoring system at 24 h post reperfusion. The area of brain infarction was inspected using 2,3,5-triphenyltetrazolium chloride staining, and the water content of brain tissue was determined by the dry-wet weight method. The morphology of neurons in the CA1 region of the rat hippocampus was inspected using Nissl staining, while the apoptosis of neurons in this region was detected by terminal-deoxynucleotidyl transferase mediated nick end labeling staining. The Sphk1 and S1P protein levels were determined by immunofluorescence and western blotting, respectively. ResultsCompared to the I/R group, rats in the Dex, PF-543, and Dex+PMA groups had a significantly lower neurological function score, as well as lower brain water content and a decreased infarction area. Moreover, the apoptotic index of the neurons and the Sphk1 and S1P levels in the hippocampal CA1 region were significantly lower in these groups (p<0.05). PMA, an agonist of Sphk1, was able to reverse the protective effects of Dex on I/R-induced neuronal cell injury. ConclusionDex could protect cerebral I/R-induced neuronal cell injury by suppressing the Sphk1/S1P signaling pathway.

Role and mechanisms of MLKL-NLRP3-mediated necroinflammation in mice with sepsis-induced acute lung injury

To investigate the roles and underlying mechanisms of mixed lineage kinase domain like (MLKL)-mediated inflammatory response induced by NOD-like receptor protein 3 (NLRP3) inflammatory corpuscles in the acute lung injury (ALI) after sepsis. Eighteen BALB/c mice were randomly divided into sham operation group (Sham group), cecal ligation and perforation (CLP)-induced sepsis model group (CLP group) and specific inhibitor Necrostatin-1 intervention group [CLP+Nec-1 group, Necrostatin-1 solution (20 mg/kg) was injected intravenously 10 minutes before modeling], with 6 mice in each group. The mice were sacrificed by neck amputation at the 2nd day after operation, and the serum and lung tissue samples were collected. The morphological changes of lung tissue were observed by hematoxylin-eosin (HE) staining. The water content of lung tissue was detected by dry-wet weight method. The pulmonary vascular permeability was measured by Evans blue (EB) staining. The protein expressions of MLKL and NLRP3 in the lung tissue were detected by Western blotting, and the level of serum interleukin-1β (IL-1β) was detected by enzyme linked immunosorbent assay (ELISA). HE staining showed that the lung morphological structure in Sham group was normal. In CLP group, congestion and edema in the alveolar cavity and interstitium, infiltration of neutrophils and thickening of alveolar wall were observed. The histopathological changes of lung tissue in CLP+Nec-1 group were better than those in CLP group. Compared with Sham group, the water content of lung tissue [(88.00±0.00)% vs. (78.00±0.01)%], pulmonary vascular permeability [EB content (mg/L): 11.82±1.15 vs. 4.00±0.71], the protein expressions of phosphorylated MLKL (p-MLKL) and NLRP3 in lung tissue (p-MLKL/GAPDH: 0.34±0.04 vs. 0.12±0.01,NLRP3/GAPDH: 0.47±0.07 vs. 0.16±0.04), and the level of serum IL-1β (ng/L: 183.56±9.61 vs. 44.14±6.95) in CLP group were all significantly increased (all P < 0.01). Compared with CLP group, the water content of lung tissue [(81.00±0.01)% vs. (88.00±0.00)%], pulmonary vascular permeability [EB content (mg/L): 7.90±0.00 vs. 11.82±1.15], protein expressions of p-MLKL and NLRP3 in lung tissue (p-MLKL/GAPDH: 0.13±0.03 vs. 0.34±0.04, NLRP3/GAPDH: 0.18±0.04 vs. 0.47±0.07), and the level of serum IL-1β (ng/L: 113.81±6.62 vs. 183.56±9.61) were all significantly decreased (all P < 0.01). MLKL-NLRP3-mediated necroinflammation was significantly up-regulatedin the lung tissue of septic mice, which could be attenuated by specific inhibitor Necrostatin-1.

Tissue sample analysis for post mortem determination of brain edema

The post mortem evaluation of a brain edema is routinely performed by pathologists based on the macroscopic signs during autopsy. This method represents the current gold standard, but is subjective and observer dependent. Therefore, three post mortem evaluation methods of brain samples were analyzed in this work: histology, wet-dry weight and normalized cerebral weight, which was described in 2020 by Bauer et al.Tissue samples from six different regions of 34 brains were collected and examined both by rating of histological slides and by measuring the water content by using a drying oven. The rating of the histological slides, stained with hematoxylin and eosin, was performed by two pathologists independently. For the water content, the wet weight and the dry weight of each sample were set in relation. The normalized cerebral weight was calculated by dividing the brain weight by the brain volume, which were both determined during autopsy and in computed tomography images, respectively.A fair to moderate interrater agreement was obtained for the histologic evaluation and a significant correlation was present between one rater and the wet-dry weight and the normalized cerebral weight method. When classifying according to the gold standard, a significant difference was detected between the edematous and nonedematous cases by using the wet-dry weight method in the cerebral cortex and by using the normalized cerebral weight method. However, the significant correlations and group differences were limited to the aforementioned results.In conclusion, both the histological and the wet-dry weight method show limited benefits for the classification of brain edema and the histology analysis is highly observer dependent. The normalized cerebral weight method, however, reveals a significant effect between the edematous and nonedematous cases when classifying according to the gold standard. Therefore, we suggest to apply this method for the assessment of brain edema since it is objective and rater independent. Nevertheless, the exact evaluation of brain edema remains a challenging task, especially due to the continuous transition between no edema and edema.

Kellerin from Ferula sinkiangensis exerts neuroprotective effects after focal cerebral ischemia in rats by inhibiting microglia-mediated inflammatory responses

Ethnopharmacological relevanceFerula sinkiangensis K. M. Shen is a traditional Chinese medicine that has a variety of pharmacological properties relevant to neurological disorders and inflammations. Kellerin, a novel compound extracted from Ferula sinkiangensis, exerts a strong anti-neuroinflammatory effect by inhibiting microglial activation. Microglial activation plays a vital role in ischemia-induced brain injury. However, the potential therapeutic effect of kellerin on focal cerebral ischemia is still unknown. Aim of the studyTo explore the effect of kellerin on cerebral ischemia and clarify its possible mechanisms, we applied the middle cerebral artery occlusion (MCAO) model and the LPS-activated microglia model in our study. Materials and methodsNeurological outcome was examined according to a 4-tiered grading system. Brain infarct size was measured using TTC staining. Brain edema was calculated using the wet weight minus dry weight method. Neuron damage and microglial activation were observed by immunofluorescence in MCAO model in rats. In in vitro studies, microglial activation was examined by flow cytometry and the viability of neuronal cells cultured in microglia-conditioned medium was measured using MTT assay. The levels of pro-inflammatory cytokines were measured by qRT-PCR and ELISA. The proteins involved in NF-κB signaling pathway were determined by western blot. Intracellular ROS was examined using DCFH-DA method and NADPH oxidase activity was measured using the NBT assay. ResultsWe found that kellerin improved neurological outcome, reduced brain infarct size and decreased brain edema in MCAO model in rats. Under the pathologic conditions of focal cerebral ischemia, kellerin alleviated neuron damage and inhibited microglial activation. Moreover, in in vitro studies of LPS-stimulated BV2 cells kellerin protected neuronal cells from being damaged by inhibiting microglial activation. Kellerin also reduced the levels of pro-inflammatory cytokines, suppressed the NF-κB signaling pathway, and decreased ROS generation and NADPH oxidase activity. ConclusionsOur discoveries reveal that the neuroprotective effects of kellerin may largely depend on its inhibitory effect on microglial activation. This suggests that kellerin could serve as a novel anti-inflammatory agent which may have therapeutic effects in ischemic stroke.

Licoricidin improves neurological dysfunction after traumatic brain injury in mice via regulating FoxO3/Wnt/β-catenin pathway.

Traumatic brain injury (TBI) is a major cause of death and disability around the world with no effective treatments currently. The present study was aimed to investigate the neuroprotective effect of licoricidin, one of the major components of licorice extract, on TBI mice and further explore the underlying mechanism. Male C57BL/6 mice were modeled by a modified weight-drop method to mimic TBI. All animals received treatment 30min after TBI. The modified Neurological Severity Score (NSS) tests were performed at 2h and 1-3days after TBI. The brain edema was analyzed by dry-wet weight method. The malonaldehyde (MDA) levels and the activities of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD) and catalase (CAT) were determined by Elisa. Apoptotic neurons were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) immunofluorescence and the expression of apoptotic proteins were measured by western blot. Activation of the FoxO3/Wnt/β-catenin was evaluated by western blot. The results showed that treatment with licoricidin could significantly decline the NSS scores and reduce the brain edema, hence promote the recovery of neurological function in TBI mice. It also elevated the phosphorylation of p66shc, brought down the levels of MDA, as well as antagonized the decrement in activities of GSH-PX, SOD and CAT induced by TBI. Moreover, licoricidin decreased the TUNEL positive neurons, downregulated the expression of Cyt-C, cleaved-Caspase-3, cleaved-Caspase-9 and Bax and upregulated the Bcl-2, attenuated cellular apoptosis. Licoricidin decreased the expression of FoxO3 and increased the Wnt/β-catenin in TBI mice. In conclusion, Licoricidin exerted neuroprotective effect on TBI model and the effect was possibly due to its antioxidative effect and antiapoptotic effect via regulating the FoxO3/Wnt/β-catenin pathway. Licoricidin may be a candidate drug for TBI therapy.

Mechanism of Fisetin Inhibiting Oxidative Stress to Alleviate the Damage of Blood Brain Barrier, Around the Intracerebral Hemorrhag Lesions

Background: The paper determined the brain water content, blood-brain barrier permeability, oxidative stress-related indicators, expressions of tight junction-associated proteins ZO-1 and Occludin, as well as expressions of ERK, p38, and JNK proteins in cerebral hemorrhage rats. The paper explored whether Fisetin reduced the damage of blood-brain barrier caused by cerebral hemorrhage through inhibiting oxidative stress, and investigated whether MAPK pathway is the main pathway for BBB damage caused by oxidative stress. Material and Methods: Rat ICH model was established. After 72 hours, mNSS score was used to measure the behavior of rats. The water content of brain tissue was determined by dry-wet weight method. Evans blue method was applied to measure the blood-brain barrier permeability. Oxidative stress index was detected by spectrophotometer. The expressions of ZO-1, Occludin, ERK, p38 and JNK proteins in rat brain tissue were determined by Western blot. Results: Fisetin significantly reduced the mNSS score, brain water content, bloodbrain barrier permeability, oxidative stress index and the expressions of ERK, p38 and JNK in rat ICH model, and also significantly increased the antioxidant index of rat ICH model, as well as the expressions of ZO-1 and Occludin. The effect is becoming more significant as the therapeutic dose is increased. Conclusion: Fisetin can inhibit the oxidative stress and alleviate the damage of BBB around the lesions of rat ICH model, thus alleviating secondary brain injury. In the rat ICH model, the expressions of ERK, p38 and JNK in the brain tissue around the lesion are increased and oxidized. The stress response is enhanced, and Fisetin inhibits oxidative stress and the expressions of ERK, p38, and JNK. MAPK is imperative in the process of oxidative stress-induced BBB damage in rat ICH model.

Effect of bloodletting acupuncture at twelve jing-well points of hand on microcirculatory disturbance in mice with traumatic brain injury

To observe the effect of bloodletting acupuncture at twelve jing-well points of hand on microcirculatory disturbance in mice with traumatic brain injury (TBI), and to explore the protective effect of bloodletting therapy on TBI. Sixty clean adult male C57BL/6J mice were randomly divided into a sham-operation group, a model group and a treatment group, 20 mice in each group. The TBI model was established by using electronic controlled cerebral cortex impact instrument in the model group and the treatment group. The mice in the treatment group were treated with bloodletting acupuncture at bilateral "Shaoshang" (LU 11), "Shangyang" (LI 1), "Zhongchong" (PC 9), "Guanchong" (TE 1), "Shaochong" (HT 9) and "Shaoze" (SI 1) immediately after trauma. The mice in the sham-operation group only opened the bone window but did not receive the strike. The regional cerebral blood flow (rCBF) was monitored by laser speckle contrast analysis (LASCA) using a PeriCam PSI System before trauma, immediately after trauma and 1, 2, 12, 24, 48, 72 h after trauma. The brain water content was measured by wet-dry weight method 24 h after trauma. The severity of functional impairment at 2, 12, 24, 48 and 72 h after trauma was evaluated by modified neurological scale scores (mNSS). ① 2 h after trauma, the mNSS in the model group and treatment group were >7 points, suggesting the successful establishment of model; compared with the sham-operation group, the mNSS was increased significantly from 12 to 72 h after trauma in the model group ( all P<0.01), but the mNSS in the treatment group was significantly lower than that in the model group from 2 to 24 h after trauma (P<0.01, P<0.05). ② Compared with the sham-operation group, rCBF in the model group was decreased significantly immediately after trauma (P<0.01), and the rCBF in the model group was lower than that in the sham-operation group from 1 to 72 h after trauma ( all P<0.01); rCBF in the treatment group began to rise and was significantly higher than that in the model group 1-2 h after trauma (P<0.01); 12-48 h after trauma, the increasing of rCBF in the two groups tended to be gentle until 72 h after injury, and rCBF in the model group was decreased while that in the treatment group continued to rise and was higher than that in the model group (P<0.01). ③ 24 h after trauma, the brain water content in the model group was significantly higher than that in the sham-operation group (P<0.01), and brain water content in the treatment group was significantly lower than that in the model group (P<0.01). The bloodletting acupuncture at twelve jing-well points of hand could improve microcirculation disturbance, increase microcirculation perfusion, alleviate secondary brain edema and promote the recovery of nerve function in mice with TBI.

Early Axonal Injury and Delayed Cytotoxic Cerebral Edema are Associated with Microglial Activation in a Mouse Model of Sepsis.

Sepsis-induced brain injury is associated with an acute deterioration of mental status resulting in cognitive impairment and acquisition of new functional limitations in sepsis survivors. However, the exact nature of brain injury in this setting is often subtle and remains to be fully characterized both in preclinical studies and at the bedside. Given the translation potential for the use of magnetic resonance imaging (MRI) to define sepsis-induced brain injury, we sought to determine and correlate the cellular changes with neuroradiographic presentations in a classic murine model of sepsis induced by cecal ligation and puncture (CLP). Sepsis was induced in 6-10-week-old male C57/BL6 mice by CLP. We used immunohistochemistry (IHC) to define neuropathology in a mouse model of sepsis along with parallel studies using MRI, focusing on cerebral edema, blood-brain barrier (BBB) disruption, and microglial activation on days 1 and 4 days after CLP. We demonstrate that septic mice had evidence of early axonal injury, inflammation, and robust microglial activation on day 1 followed by cytotoxic edema on day 4 in the cortex, thalamus, and hippocampus in the absence of BBB disruption. We note the superiority of the MRI to detect subtle brain injury and cytotoxic cerebral edema in comparison with the traditional gold standard assessment, i.e., percent brain water (wet-dry weight method). We conclude that inflammatory changes in the septic brain can be detected in real time, and further studies are needed to understand axonal injury and the impact of inhibition of microglial activation on the development of cerebral edema.

Calcitriol alleviates global cerebral ischemia-induced cognitive impairment by reducing apoptosis regulated by VDR/ERK signaling pathway in rat hippocampus

BackgroundVitamin D (VD) has important neuroprotective functions in the central nervous system. However, further exploration is still needed in the neuroprotective effects of VD monomer therapy on global cerebral ischemia (GCI) and its potential molecular mechanism. ObjectiveTo investigate whether calcitriol, a biologically active metabolite of VD, could alleviate cognitive impairment induced by GCI via reducing cell apoptosis and activating the extracellular signal-regulated kinase (ERK) signaling pathway. MethodsA total of 145 adult male Sprague Dawley rats were randomly divided into five groups: Sham group (n = 45), GCI group (n = 45), calcitriol treatment group (GCI + calcitriol, n = 45), PD98059 treatment group (n = 5) and vehicle group (n = 5). Morris water maze test was used for evaluating spatial learning and memory functions. Neurological Severity Score and wet-dry weight method were applied to detect neurological deficits and brain water content, respectively. Hematoxylin and eosin staining, transmission electron microscopy, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling staining were performed for evaluating the changes of hippocampal CA1 neurons. Immunocytochemistry, immunofluorescence chemistry, and western blot analysis were performed for evaluating the changes of related proteins. ResultsCalcitriol significantly ameliorated the spatial learning and memory impairments, improved neurological function, attenuated brain edema, and improved the morphological defects in the CA1 area of the hippocampus. Besides, calcitriol reduced GCI-induced cell apoptosis and reversed the up-regulation of pro-apoptotic proteins (Caspase-3 and Bax) and the down-regulation of anti-apoptotic protein (Bcl-2). Furthermore, calcitriol also increased the expression of VD receptors (VDR) and activated the ERK signaling pathway. Moreover, the p-ERK1/2 inhibitor PD98059 reversed the effect of calcitriol on the expression of apoptosis-related proteins. ConclusionsCalcitriol may have a protective effect against GCI-induced cognitive impairments via inhibition of apoptotic cascade by activating the VDR/ERK signaling pathway.

Effect of dexmedetomidine combined with hypothermia on acute brain edema in mice with traumatic brain injury

Objective To explore the effect of Dexmedetomidine (Dex) on acute brain edema in mice in condition with targeted temperature management (TTM) following traumatic brain injury (TBI). Methods A total of 180 male C57BL/6J mice were divided into control group, sham operation group, TBI group, TBI+ Dex group, TBI+ TTM group, and TBI+ Dex+ TTM group according to the random number table (n=30 per group). The sham operation group only opened the bone window but did not hit it, and the control group did not open the bone window. The TBI+ Dex, TBI+ TTM, and TBI+ Dex+ TTM groups were intraperitoneally injected with Dex (60 μg/kg once every 2 h for 3 times) and/or hypothermia after TBI. The brain tissue injury volume, EB extravasation and brain water content of each group were determined by toluidine blue, Evans blue staining and dry-wet weight method at 24 hours after injury. Real-time quantitative PCR and Western blot were used to detect the expression of Claudin-5 in the injured brain tissue. At 24, 48, and 72 hours after injury, the neurological deficiency degree was assessed using the modified neurological severity scores(mNSS). Results Compared with the sham operation group, TBI mice showed significant increase in brain tissue injury volume [(0.49±0.04)mm3vs. (11.57±1.01)mm3], blood-brain barrier permeability [(16.4±0.8)μg/g vs. (54.3±1.7)μg/g], brain tissue water content [(76.7±0.9)% vs. (83.1±0.8)%], and mNSS score [(1.6±0.7)points vs. (13.4±0.7)points] at 24 hour after TBI (all P<0.01). However, Dex or TTM treatment reduced brain tissue injury volume [(7.20±0.18)mm3 and (5.94±0.18)mm3], blood-brain barrier permeability [(32.7±1.2)μg/g and (27.6±1.0)μg/g], brain tissue water content [(78.5±0.4)% and (78.2±0.6)%], and neurological function [mNSS: (7.3±1.1)points and (5.8±1.3)points] (all P<0.01). Moreover, Dex+ TTM group showed better neuroprotection [reduced brain tissue injury volume: (3.92±0.05)mm3, reduced BBB permeability: (21.6±0.7)μg/g, reduced brain water content: (77.7±0.3)%, and reduced mNSS: (4.3±1.2)points] compared with Dex or TTM alone (all P<0.01). Additionally, the mRNA expression of Claudin-5 (0.23±0.01) decreased significantly at 24 hours after TBI compared with sham group (0.93±0.04, P<0.01), but Dex or TTM could increase the expression of Claudin-5 (0.47±0.01, and 0.54±0.09) compared with TBI group (P<0.01), especially that of TBI+ Dex+ TTM group (0.64±0.02, P<0.01). Furthermore, the protein expression of Claudin-5 was in accordance with the result of its mRNA expression. Conclusion Dex in condition with targeted temperature management can up-regulate Claudin-5 expression in early TBI, protect the integrity of blood-brain barrier, attenuate acute brain edema and neurological damage, and improve neurological function recovery. Key words: Dexmedetomidine; Brain injuries; Brain edema; Hypothermia; Claudin-5

RETRACTED: Repression of long non-coding RNA MEG3 restores nerve growth and alleviates neurological impairment after cerebral ischemia-reperfusion injury in a rat model

This study was performed to investigate effect of long non-coding RNA (lncRNA) MEG3 on nerve growth and neurological impairment in a rat model after cerebral ischemia-reperfusion injury (IRI) through the Wnt/β-catenin signaling pathway. Rat models of middle cerebral artery occlusion (MCAO) were established to stimulate an environment of cerebral IRI. The modeled rats were subjected to negative control (NC), MEG3, si-MEG3, classical Wnt pathway inhibitor DKK1 or classical Wnt pathway activator LiCl to validate the effect of MEG3 on neurological impairment and nerve growth. Neurological deficit scoring, fault-foot test and balance beam test were performed to assess neurological impairment. TTC staining, dry-wet weight method and Evan's blue (EB) staining were employed to determine infarct area, water content of brain tissues and blood-brain barrier (BBB) permeability, respectively. Neuronal apoptosis and necrosis were observed by TUNEL staining and Fluoro-Jade C staining. ELISA was adopted to identify levels of nerve growth factors to identify neurogenesis conditions, including brain derived neurotrophic factor (BDNF), nerve growth factor (NGF) and basic fibroblast growth factor (bFGF). Nissl staining was used to detect the survival of neurons in brain tissues of rats. Western blot analysis was used to detect the expression of key proteins in Wnt/β-catenin signaling pathway in brain tissues. High expression of MEG3 was identified in rat models of MACAO, the brain tissues of which manifested obvious neurological impairment, increased infarct area, water content, BBB permeability, accelerated neuronal apoptosis and necrosis, increased surviving neurons, upregulated expression of key proteins in Wnt/β-catenin signaling pathway and elevated levels of BDNF, NGF and bFGF. With the treatment of si-MEG3, the MEG3 expression was reduced; whereby, modeled rats showed ameliorated neurological impairment, reduced infarct area, water content, BBB permeability, neuronal apoptosis and necrosis and significantly enhanced neurogenesis. The treatment of MEG3 exhibited an opposite trend. After treatment with DKK1, the effect of si-MEG3 was reversed. After treatment with LiCl, the effect of MEG3 was reversed. Based on the findings of this study, down-regulation of lncRNA MEG3 expression enhanced nerve growth and alleviated neurological impairment of rats after cerebral IRI through the activation of the Wnt/β-catenin signaling pathway.

Therapeutic hypothermia reduced brain damage on rats after cardiopulmonary resuscitation by activating III-type PI3K pathway to increase autophagy

To investigate the effect of III-type phosphatidylinositide 3 kinase (PI3K) pathway adjusting autophagy on brain damage mechanism after cardiopulmonary resuscitation (CPR) and hypothermia treatment. The asphyxia induce cardiac arrest-CPR model was reproduced on healthy male Sprague-Dawley (SD) rats. Sixty rats after restoration of spontaneous circulation (ROSC) were randomly divided into normothermia group, therapeutic hypothermia group and PI3K inhibitor 3-methyl adenine (3-MA) pretreatment group, differentiated by 24 hours and 48 hours after ROSC. Each group had 10 rats at each time point. The anal temperature in the normothermia group was maintained at (37.0±0.2) centigrade, and the rats in the hypothermia group were given cooling treatment immediately after ROSC, and the target rectal temperature was 32-34 centigrade. In the 3-MA pretreatment group, 10 mmol/L 3-MA 5 μL was injected into the ventricle 20 minutes before asphyxia, and other groups were given the same amount of normal saline. Ten rats without CPR were included in Sham group only received anesthesia and catheterization. The rats were sacrificed at 24 hours and 48 hours after ROSC respectively, and the brain tissues were harvested, the brain water content (BWC) was measured by dry-wet weight method. Western Blot was used to determine the autophagy related proteins Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3), apoptosis related proteins Bcl-2 and caspase-3, and the III-type PI3K pathway proteins Vps34 and Atg14. Ultrastructural changes in brain tissue were observed with transmission electron microscope. Neurological deficit scores (NDS) was obtained in each group at 48 hours after ROSC. Compared with Sham group, the cortex at 24 hours after ROSC in normothermic group showed obvious edema, apoptosis and autophagy began to appear under transmission electron microscope, and the expressions of autophagy, apoptosis and III- type PI3K-related proteins in brain tissue were significantly increased in a time-dependent manner, and the neurological function at 48 hours after ROSC was significantly damaged. After hypothermia intervention, brain edema of rats was significantly reduced, no obvious apoptosis was found, but autophagy was increased, the expressions of autophagy-related proteins Vps34, Atg14 and III-type PI3K-related proteins Beclin-1 and LC3 at 48 hours after ROSC were further higher than those of normothermic group (Vps34/GAPDH: 0.25±0.03 vs. 0.15±0.04, Atg14/GAPDH: 0.12±0.03 vs. 0.05±0.04, Beclin-1/GAPDH: 0.060±0.002 vs. 0.018±0.002, LC3 -II/GAPDH: 0.160±0.010 vs. 0.050±0.010, all P < 0.05), the expressions of apoptosis related proteins Bcl-2 and caspase-3 were significantly lowered (Bcl-2/GAPDH: 0.05±0.03 vs. 0.20±0.04, caspase-3/GAPDH: 0.050±0.002 vs. 0.140±0.015, both P < 0.05), neurological function was significantly improved (NDS: 157±85 vs. 343±198, P < 0.05). Pretreatment with 3-MA inhibited the protective effect of hypothermia on brain tissues. The expressions of Vps34, Atg14, Beclin-1 and LC3 in brain tissues at 48 hours after ROSC in 3-MA pretreatment group was significantly lower than those in the hypothermia group (Vps34/GAPDH: 0.18±0.03 vs. 0.25±0.03, Atg14/GAPDH: 0.07±0.04 vs. 0.12±0.03, Beclin-1/GAPDH: 0.015±0.003 vs. 0.060±0.002, LC3-II/GAPDH: 0.045±0.030 vs. 0.160±0.010, all P < 0.05), the expressions of Bcl-2 and caspase-3 were significantly increased (Bcl-2/GAPDH: 0.15±0.04 vs. 0.05±0.03, caspase-3/GAPDH: 0.120±0.015 vs. 0.050±0.002, both P < 0.05), and NDS score was significantly increased (341±208 vs. 157±85, P < 0.05). Hypothermia treatment reduced brain edema and ameliorated brain function after CPR, which might be related to increase autophagy and inhibit apoptosis adjustment by activating III-type PI3K pathway.

Neuroprotective Efficiency of Cyclosporine After Traumatic Brain Injury in Rats.

To evaluate the possible neuroprotective effects of systemic administration of cyclosporine (Cyclosporin A) after traumatic brain injury in rats. The modified Feeney method was used as the trauma model in male Sprague Dawley rats. After the trauma, 20 mg/kg of cyclosporine was administered to the one group of the rats (n=12) intraperitoneally. Twenty-four hours after injury, the subjects were sacrificed, and brain samples were removed. The level of brain edema was evaluated through the wet-dry weight method, the lipid peroxidation ratio, and histological examination by transmission electron microscopy. The level of brain edema and lipid peroxidation ratio significantly decreased in the rats that received cyclosporine. Ultrastructural neurodestruction was graded, and a comparison of the scores between the experimental groups revealed significant neuroprotective effects of cyclosporine. The results demonstrated that systemic administration of cyclosporine produces a statistically significant decrease in both the level of brain edema and lipid peroxidation ratio when compared with "no treatment". Cyclosporine, which is regularly used as an immunosuppressant agent, is also known to prevent opening of the mitochondrial permeability transition pore by unbinding mitochondrial matrix cyclophilin. Regulation of transition pore for mitochondrial permeability by cyclosporine implies that mitochondrial dysfunction following traumatic brain injury is an important event in the progressive loss of neuronal tissue.

Proton Magnetic Resonance Spectroscopy (H1-MRS) Study of the Ketogenic Diet on Repetitive Mild Traumatic Brain Injury in Adolescent Rats and Its Effect on Neurodegeneration

A ketogenic diet (KD) improves cellular metabolism and functional recovery after moderate-to-severe traumatic brain injury. Here, we evaluated the changes of neurochemical metabolites after KD therapy for repetitive mild traumatic brain injury (rmTBI) and its possible role in neurodegeneration. Postnatal day 35 rats were randomly divided into 3 groups: sham, control, and KD groups. Rats in control and KD groups were given 3 rmTBI by a fluid percussion traumatic brain injury device 24 hours apart. All rats were killed at 7 days after the last injury. The ipsilateral cortex were analyzed with hematoxylin and eosin staining; beta-hydroxybutyrate was measured; conventional magnetic resonance imaging and the dry-wet weight method were used to assess the brain edema; changes of neurochemical metabolites were assessed using the ratio of N-acetylaspartate (NAA)/creatine (Cr), choline compound (Cho)/Cr, and NAA/Cho with magnetic resonance spectroscopy; the effect of KD therapy on neurodegeneration was evaluated with double immunofluorescence staining of Iba-1/beclin-1; behavioral outcome was assessed with beam walk/beam balance tests. KD significantly elevated beta-hydroxybutyrate levels, and there was no brain edema associated with rmTBI and KD therapy; behavioral assessment showed KD therapy significantly improved motor performance; magnetic resonance spectroscopy showed that rmTBI reduced the ratio of NAA/Cr and had no effect on the ratios of Cho/Cr and NAA/Cho whereas KD increased the ratio of NAA/Cr; double immunofluorescence staining showed KD therapy could significantly decrease microglial beclin-1 expression in the ipsilateral cortex. These results suggest the effect of KD on metabolic status and its possible role in preventing neurodegeneration in adolescent rats after rmTBI.