Inhibitory effect on cerebral inflammatory agents that accompany traumatic brain injury in a rat model: a potential neuroprotective mechanism of recombinant human erythropoietin (rhEPO)

Erythropoietin (EPO) has recently been shown to have a neuroprotective effect in animal models of traumatic brain injury (TBI). However, the precise mechanisms remain unclear. Cerebral inflammation plays an important role in the pathogenesis of secondary brain injury after TBI. We, therefore, tried to analyze how recombinant human erythropoietin (rhEPO) might effect the inflammation-related factors common to TBI: nuclear factor kappa B (NF-kappaB), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in a rat TBI model. Male rats were given 0 or 5000 units/kg injections of rhEPO 1h post-injury and on days 1, 2 and 3 after surgery. Brain samples were extracted at 3 days after trauma. We measured NF-kappaB by electrophoretic mobility shift assay (EMSA); IL-1beta, TNF-alpha and IL-6 by enzyme-linked immunosorbent assay (ELISA); ICAM-1 by immunohistochemistry; brain edema by wet/dry method; blood-brain barrier (BBB) permeability by Evans blue extravasation and cortical apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. We found that NF-kappaB, pro-inflammatory cytokines and ICAM-1 were increased in all injured animals. In animals given rhEPO post-TBI, NF-kappaB, IL-1beta, TNF-alpha and ICAM-1 were decreased in comparison to vehicle-treated animals. Measures of IL-6 showed no change after rhEPO treatment. Administration of rhEPO reduced brain edema, BBB permeability and apoptotic cells in the injured brain. In conclusion, post-TBI rhEPO administration may attenuate inflammatory response in the injured rat brain, and this may be one mechanism by which rhEPO improves outcome following TBI.     

Intracranial injection of recombinant stromal-derived factor-1 alpha (SDF-1α) attenuates traumatic brain injury in rats

Objective

This study was conducted to investigate the role of stromal-derived factor-1 alpha (SDF-1α) in a secondary brain injury after traumatic brain injury (TBI) in rats, and to further elucidate its underlying regulatory mechanisms.

Materials and methods

Male Sprague–Dawley rats underwent TBI for 30 min, and then received intracranial injections of recombinant SDF-1α, SDF-1α antibody, or saline as a vehicle control. At 24 h after TBI, brain tissues from the experimental animals were subjected to histology, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and western blot analyses.

Results

TBI-induced brain edema and blood–brain barrier disruption were ameliorated by post-injury injections of SDF-1α. TBI-induced neuronal degradation and apoptosis, accompanied by increased cleaved caspase-3, cleaved PARP and Bax, and decreased Bcl-2 were found to be attenuated by SDF-1α injection. Nitric oxide (NO) and inducible nitric oxide synthase (iNOS) levels decreased in SDF-1α treated animals after TBI. SDF-1α repressed inflammatory responses by inhibiting the expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. However, neutralizing the effect of SDF-1α with its antibody abolished these therapeutic alterations in TBI animals. Importantly, SDF-1α attenuated the brain lesion by affecting the ERK and NF-κB signaling pathways after mechanical head trauma in rats.

Conclusions

SDF-1α ameliorates mechanical trauma-induced pathological changes via its anti-apoptotic and anti-inflammatory action in the brain.     

Attenuation of interleukin-1beta by pulsed electromagnetic fields after traumatic brain injury

Traumatic Brain Injury (TBI) is a major cause of morbidity and mortality in civilian and military populations. Interleukin-1beta (IL-1β) is a pro-inflammatory cytokine with a key role in the inflammatory response following TBI and studies indicate that attenuation of this cytokine improves behavioral outcomes. Pulsed electromagnetic fields (PEMF) can reduce inflammation after soft tissue injuries in animals and humans. Therefore, we explored whether PEMF signals could alter the course of IL-1β production in rats subjected to closed-head contusive weight-drop injuries (Marmarou method) and penetrating needle-stick brain injuries. Protein levels, measured by the Biorad assay, were not altered by injuries or PEMF treatment. In addition, we verified that IL-1β levels in cerebrospinal fluid (CSF) were proportional to injury severity in the contusion model. Results demonstrate that PEMF treatment attenuated IL-1β levels up to 10-fold in CSF within 6 h after contusive injury and also significantly suppressed IL-1β within 17–24 h after penetrating injury. In contrast, no differences in IL-1β were seen between PEMF-treated and control groups in brain homogenates. To the authors’ knowledge, this is the first report of the use of PEMF to modulate an inflammatory cytokine after TBI. These results warrant further studies to assess the effects of PEMF on other inflammatory markers and functional outcomes.     

Delayed glucose treatment improves cognitive function following fluid-percussion injury

Experimental traumatic brain injury (TBI) results in marked neurochemical and metabolic changes. Research has demonstrated that after the initial insult the brain undergoes an immediate state of hypermetabolism followed by a sustained period of hypometabolism. The altered extra- and intracellular environment can compromise neuronal performance and limit functional recovery. If brain metabolism is depressed chronically after TBI, then interventions that are designed to increase metabolism may be beneficial to outcome. Glucose treatment has been shown to improve cognition in many populations, particularly those with cognitive deficits. The following experiments examined the effects of delayed postinjury glucose supplementation on cognitive function following TBI. Male Sprague-Dawley rats received either sham or lateral fluid-percussion (LFP) injury. Cognitive functioning was assessed with the Morris water maze (MWM) on postinjury days 11-15. In the first experiment, saline or 100mg/kg glucose was administered 10 min before cognition assessment. Injured animals treated with glucose displayed significantly shorter latencies to reach the goal platform compared to injured saline-treated animals. Glucose had no effect on sham-injured rats. In the second experiment, injured rats were given daily injections of saline or 100mg/kg glucose for 10 days beginning 24h after injury. Rats were then tested in the MWM on days 11-15 without glucose or saline treatment. In this experiment, glucose treatment did not affect MWM performance. These data provide evidence that the chronic energy supplementation after TBI improves outcome when administered shortly before cognitive assessment.     

孕酮抑制大鼠脑损伤后炎症反应诱导的神经细胞凋亡

目的:研究孕酮(progesterone,PROG)对大鼠脑损伤后皮质COX-2、iNOS、NF-κB和caspase-3表达的影响,以探讨PROG脑保护作用的可能机制。方法:雄性SD大鼠45只,随机分为假手术组、脑损伤组和PROG治疗组,按照改良的Feeney自由落体损伤装置制作大鼠脑损伤模型。PROG治疗组伤后1,6h腹腔注射PROG16mg/kg,各组于伤后24h取材。用免疫组织化学法及免疫蛋白印迹法,观察大鼠皮质COX-2、iNOS、NF-κB和caspase-3的免疫阳性细胞数和蛋白表达水平变化。结果:与假手术组大鼠相比,脑损伤组大鼠皮质COX-2、iN-OS、NF-κB和caspase-3阳性细胞数和蛋白表达水平较假手术组COX-2、iNOS、NF-κB和caspase-3显著增加(P0.05);PROG治疗组与脑损伤组比较,大鼠皮质COX-2、iNOS、NF-κB和caspase-3阳性细胞数和蛋白表达水平明显减少(P0.05)。结论:PROG可通过抑制NF-κB的活化,阻断NF-κB和iNOS自身强化的炎症反应过程,降低COX-2、caspase-3的表达,抑制脑损伤后的炎症反应和细胞凋亡,从而发挥脑保护作用。     

Intranasal administration of progesterone increases dopaminergic activity in amygdala and neostriatum of male rats

We evaluated the effects of intranasal administration of progesterone (PROG) on the activity of dopaminergic neurons in the brain of anesthetized rats by means of microdialysis. Male Wistar rats were implanted with guide cannulae in the basolateral amygdala and neostriatum. Three to 5 days later, they were anesthetized with urethane, and dialysis probes were inserted. After a stabilization period of 2 h, four 30-min samples were collected. Thereafter, the treatment (0.5, 1.0 or 2.0 mg/kg of PROG dissolved in a viscous castor oil mixture, or vehicle) was applied into the nose in a volume of 10 microl (5 microl in each nostril). In other animals, an s.c. injection of PROG (1.0, 2.0 or 4.0 mg/kg) or vehicle was given. Samples of both application ways were collected at 30-min interval for 4 h after the treatment and immediately analyzed with high performance liquid chromatography and electrochemical detection. Intranasal administration of 2 mg/kg of PROG led to an immediate (within 30 min after the treatment) significant increase in the basolateral amygdala dopamine levels. In the neostriatum, the 2 mg/kg dose led to a delayed significant increase in dopamine. S.c. administration of 4 mg/kg of PROG was followed by a delayed significant increase in dopamine, both, in the basolateral amygdala and neostriatum, but smaller in magnitude in comparison to the intranasal treatment. This is the first study to demonstrate dopamine-enhancing effects of PROG, not only in the neostriatum, but also in the basolateral amygdala. Our results indicate that the intranasal route of administration of PROG is a more efficacious way for targeting the brain than the s.c. route.     

Nicotinamide reduces acute cortical neuronal death and edema in the traumatically injured brain

Previous studies have shown that administration of nicotinamide (Vitamin B(3)) in animal models of traumatic brain injury (TBI) and ischemia significantly reduced the size of infarction or injury and improved functional recovery. The present study evaluated the ability of nicotinamide to provide acute neuroprotection and edema reduction following TBI. Groups of rats were assigned to nicotinamide (500mg/kg) or saline (1.0ml/kg) treatment conditions and received contusion injuries or sham surgeries. Drug treatment was administered 15min following injury. Brains were harvested 24h later and either processed for histology or water content. Frozen sections were stained with the degenerating neuron stain (Fluoro-Jade B) (FJ) and cell counts were performed at the site of injury. Additional brains were processed for water content (a measure of injury-induced edema). Results of this study showed that administration of nicotinamide following TBI significantly reduced the number of FJ(+) neurons in the injured cortex compared to saline-treated animals. Examination of the water content of the brains also revealed that administration of nicotinamide significantly attenuated the amount of water compared to saline-treated animals in the injured cortex. These results indicate that nicotinamide administration significantly reduced neuronal death and attenuated cerebral edema following injury. The current findings suggest that nicotinamide significantly modulates acute pathophysiological processes following injury and that this may account for its beneficial effects on recovery of function following injury.     

人脂肪间充质干细胞来源的外排体促进大鼠创伤性脑损伤后神经功能恢复

目的研究人脂肪间充质干细胞(h AMSCs)来源的外排体对创伤性脑损伤(TBI)的治疗作用及其可能的机制。方法分离健康成人脂肪MSCs,通过超滤法提取外排体。将大鼠分成:假手术组,PBS对照组,MSC治疗组,exosomes治疗组。于TBI建模24 h后,治疗组分别沿损伤边缘区局部注射,PBS 30μL,MSC 2×10~5个细胞/只,exosomes 25μg总蛋白量/只,总体积30μL。在建模前和TBI后1、3、7、10、13、16和30 d测试所有大鼠的m NSS评分和前肢踩空试验。3和7d处死大鼠,提取大鼠脑组织总RNA,实时定量PCR检测大鼠炎性因子TNF-α和IL-1β的表达,30 d处死大鼠,tunel-neun双标免疫荧光检测TBI后神经元凋亡。结果外排体的治疗显著促进TBI后的神经功能的恢复,治疗效果与MSC治疗效果相当,其机制可能是通过抑制大鼠TBI后急性炎性反应,减少神经元凋亡。结论人脂肪间充质干细胞来源的外排体促进脑外伤后神经功能的恢复,这将为临床提供一种新的更安全的TBI治疗手段。     

Genetic and histologic evidence implicates role of inflammation in traumatic brain injury-induced apoptosis in the rat cerebral cortex following moderate fluid percussion injury

Traumatic brain injury (TBI) causes massive brain damage. However, the secondary injury and temporal sequence of events with multiple mechanisms after the insult has not been elucidated. Here, we examined the occurrence of apoptosis and a causal relationship between inflammation and apoptosis in the TBI brain. Following a lateral moderate fluid percussion injury model of TBI in adult rats, microarray analyses detected apparent changes in the expression levels of apoptosis-related genes which revealed time-dependent expression patterns for 23 genes in the lateral cortex. The upregulated 23 genes included inflammatory cytokines such as interleukin 1 (IL-1) α, IL-1β, and tumor necrotic factor (TNF) which immediately increased at 3 h following the injury. Time-dependent gene expression profile analyses showed that apoptosis was subsequently induced following inflammation. These results taken together suggested changes in expression of apoptosis-related genes may be associated with inflammatory response. Accompanying this surge of cell death genes after TBI was a neurostructural pathologic hallmark of apoptosis characterized by leakage of cytochrome c into cytoplasm, DNA fragmentation and apoptotic cells in the lateral cortex of the impacted hemisphere. Caspase-3 positive cells in the TBI brain were initially sporadic after 3 h, but these apoptotic cells subsequently increased and populated the cerebral cortex at 6 and 12 h, and gradually reached a plateau by 48 h. Interestingly, the expression profile of CD68 macrophage labeled cells closely resembled that of apoptotic cells after TBI, including the role of inflammatory signaling pathway in the progression of apoptotic cell death. These results taken together suggest that TBI induced upregulation of apoptosis-related genes, concomitant with the detection of apoptotic brain pathology during the 3-48 h post-injury period, which may be likely mediated by inflammation. Therapies designed at abrogating apoptosis and/or inflammation may prove effective when initiated at this subacute TBI phase.     

Progesterone exerts neuroprotective effects by inhibiting inflammatory response after stroke

Objective and design  We evaluated the inhibitory effects of progesterone (PROG) on inflammatory response and its influence on the structure of blood–brain barrier in a permanent model of stroke. Material  One hundred and twenty adult male Sprague-Dawley rats were used in this study. Treatments  PROG was dissolved in 22.5% 2-hydroxypropyl-bcyclodextrin and given in a dose of 15 mg/kg by intraperitoneal injection 1 h after permanent occlusion of middle cerebral artery (pMCAO). Additional injections of 15 mg/kg were administered subcutaneously 6, 24, and 48 h after pMCAO. Methods  The expression of tumor necrosis factor-alpha (TNF-α) and claudin5 was measured by immunohistochemistry and western blot technique. Brain water content was determined by the dry–wet weight method. Results  TNF-α were increased, but claudin5 were reduced in vehicle-treated rats after pMCAO. PROG-treated rats showed a substantial reduction in the expression of TNF-α compared to vehicle controls. In addition, there was significant increase in the expression of claudin5 in the pMCAO rats treated with PROG compared to vehicle. Examination of the water content of the brain also revealed that administration of PROG significantly attenuated the amount of water compared to vehicle in the ipsilateral hemispheres. Conclusions  These data indicate that PROG is beneficial in this animal model, and may warrant further test in future clinical trials for human stroke.     

NNZ-2566 treatment inhibits neuroinflammation and pro-inflammatory cytokine expression induced by experimental penetrating ballistic-like brain injury in rats

Background

Inflammatory cytokines play a crucial role in the pathophysiology of traumatic brain injury (TBI), exerting either deleterious effects on the progression of tissue damage or beneficial roles during recovery and repair. NNZ-2566, a synthetic analogue of the neuroprotective tripeptide Glypromate^®, has been shown to be neuroprotective in animal models of brain injury. The goal of this study was to determine the effects of NNZ-2566 on inflammatory cytokine expression and neuroinflammation induced by penetrating ballistic-like brain injury (PBBI) in rats.

Methods

NNZ-2566 or vehicle (saline) was administered intravenously as a bolus injection (10 mg/kg) at 30 min post-injury, immediately followed by a continuous infusion of NNZ-2566 (3 mg/kg/h), or equal volume of vehicle, for various durations. Inflammatory cytokine gene expression from the brain tissue of rats exposed to PBBI was evaluated using microarray, quantitative real time PCR (QRT-PCR), and enzyme-linked immunosorbent assay (ELISA) array. Histopathology of the injured brains was examined using hematoxylin and eosin (H&;E) and immunocytochemistry of inflammatory cytokine IL-1β.

Results

NNZ-2566 treatment significantly reduced injury-mediated up-regulation of IL-1β, TNF-α, E-selectin and IL-6 mRNA during the acute injury phase. ELISA cytokine array showed that NZ-2566 treatment significantly reduced levels of the pro-inflammatory cytokines IL-1β, TNF-α and IFN-γ in the injured brain, but did not affect anti-inflammatory cytokine IL-6 levels.

Conclusion

Collectively, these results suggest that the neuroprotective effects of NNZ-2566 may, in part, be functionally attributed to the compound's ability to modulate expression of multiple neuroinflammatory mediators in the injured brain.     

Functional endothelial progenitor cells derived from adipose tissue show beneficial effect on cell therapy of traumatic brain injury

Endothelial progenitor cells (EPCs) are responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Adipose tissue (AT) is an abundant source of mesenchymal stem cells (MSCs), which have multipotent differentiation ability. We successfully derived EPCs from AT, which maintained a strong proliferative capacity and demonstrated the characteristic endothelial function of uptaking of acetylated low-density lipoprotein. They formed tube-like structures in vitro. Endothelial nitric oxide synthase (eNOS) gene expression in EPCs was similar to that in mature endothelial cells. Transplantation of EPCs derived from AT after the acute phase was applied in rats with traumatic brain injury (TBI). Transplanted EPCs participated in the neovascularization of injured brain. Improving functional recovery, reducement of deficiency volume of brain, host astrogliosis and inflammation were found. These results suggest that adult AT derived stem cells can be induced to functional EPCs and have beneficial effect on cell therapy.     

The synthetic NCAM-derived peptide, FGL, modulates the transcriptional response to traumatic brain injury

Cerebral responses to traumatic brain injury (TBI) include up- and downregulation of a vast number of proteins involved in endogenous inflammatory responses and defense mechanisms developing postinjury. The present study analyzed the global gene expression profile in response to cryo-induced TBI by means of microarray analysis. Adolescent rats were subjected to TBI and treated with either placebo or a neural cell adhesion molecule (NCAM)-derived fibroblast growth factor receptor (FGFR) agonist, FGL peptide, which has been demonstrated to have neuroprotective effects. mRNA levels were measured at various time-points postlesion (6 h, 1 day and 4 days). The effects of injury, treatment, and injury-treatment interaction were observed. TBI alone rendered a large number of genes affected. Analysis of lesion and treatment interactions resulted in a clear effect of the interaction between injury and FGL-treatment compared to injury and placebo-treatment. Genes affected by TBI alone included inflammation markers, protein kinases, ion channel members and growth factors. Genes encoding regulators of apoptosis, signal transduction and metabolism were altered by the interaction between FGL-treatment and TBI. FGL-treatment in non-injured animals rendered genes regulating signaling, transport and cytoskeleton maintenance significantly increased. Thus, the hypothesis of a putative neuroprotective role of FGL was supported by our findings.     

骨髓间充质干细胞立体定向移植联合亚低温治疗大鼠脑损伤

背景：临床研究证明,亚低温(33~35 ℃)能有效减轻继发性脑和脊髓损伤,对中枢神经损伤有确切的保护作用。 目的：检测是否可以通亚低温治疗的方法提高骨髓间充质干细胞立体定向移植对重型颅脑损伤大鼠的治疗效果。 方法：采用液压颅脑损伤仪,给予Wistar大鼠2.53.31~303.98 kPa液压冲击力,制成重型液压颅脑损伤大鼠模型,将其随机分为脑损伤组,骨髓间充质干细胞移植组,亚低温+骨髓间充质干细胞组。后2组伤后6 h将体外培养的SD大鼠骨髓间充质干细胞立体定向移植到脑损伤灶内,亚低温+骨髓间充质干细胞组同时给予低温治疗。伤后第3天用Western Blot检测脑组织中AQP4蛋白合成的变化,采用干湿比重法测脑组织含水量。伤后24 h,3 d及伤后1,2 周行动物神经学缺损评分,2周后处死行免疫组织化学和苏木精-伊红染色。 结果与结论：亚低温治疗后,与脑损伤组和骨髓间充质干细胞移植组相比,亚低温+骨髓间充质干细胞组AQP4蛋白表达量及脑水肿程度也明显降低(P < 0.05),移植后1和2周,亚低温+骨髓间充质干细胞组的大鼠神经学缺损评分明显低于其他两组;且其脑组织切片中的神经元数量较其他两组明显增多(P < 0.01)。提示骨髓间充质干细胞立体定向移植联合亚低温治疗大鼠脑损伤可明显改善重型颅脑损伤后大鼠的神经学功能。     

Oral fish oil restores striatal dopamine release after traumatic brain injury

Omega-3 fatty acid administration can affect the release of neurotransmitters and reduce inflammation and oxidative stress, but its use in traumatic brain injury (TBI) has not been described extensively. We investigated the effect of 7 day oral fish oil treatment in the recovery of potassium evoked dopamine release after TBI. Sham rats and TBI rats were given either olive oil or fish oil by oral gavage and were subject to cerebral microdialysis. Olive oil treated TBI rats showed significant dopamine release deficit compared to sham rats, and this deficit was restored with oral fish oil treatment. There was no effect of fish oil treatment on extracellular levels of dopamine metabolites such as 3,4-dihydroxyphenylacetic acid and homovanillic acid. These results suggest the therapeutic potential of omega-3 fatty acids in restoring dopamine neurotransmission deficits after TBI.     

Genomic profile of Toll-like receptor pathways in traumatically brain-injured mice: effect of exogenous progesterone

Background  

Traumatic brain injury (TBI) causes acute inflammatory responses that result in an enduring cascade of secondary neuronal loss and behavioral impairments. It has been reported that progesterone (PROG) can inhibit the increase of some inflammatory cytokines and inflammation-related factors induced by TBI. Toll-like receptors (TLRs) play a critical role in the induction and regulation of immune/inflammatory responses. Therefore, in the present study, we examined the genomic profiles of TLR-mediated pathways in traumatically injured brain and PROG's effects on these genes.     

MK-801对新生鼠脑外伤性神经变性的超微结构的影响

目的:研究非竞争性N-甲基-D-天门冬氨酸受体拮抗剂MK-801对新生鼠创伤性颅脑损伤(TBI)后同侧顶叶皮质和海马神经元超微结构的影响.方法:新生7 d SD大鼠,被随机分成正常对照组和实验组(实验组给予MK-801 1 mg/kg,并且细分为创伤前30 min给药、创伤即刻给药和创伤后30 min给药3个亚组).造模24 h取材,透射电镜下观察神经细胞超微结构的变化.结果:创伤后30 min给药的神经细胞出现胞质和核染色质的浓缩深染,胞质内充满大小不等的空泡;创伤即刻组的神经细胞胞质内线粒体有肿胀,但胞核的变化不明显.结论:适时和适量运用MK-180能延迟神经细胞核染色质和粒线体的变性损伤,使之停留在损伤早期,为临床联合其他药物治疗赢得时间.     

Delayed sodium pyruvate treatment improves working memory following experimental traumatic brain injury

Prior work indicates that cerebral glycolysis is impaired following traumatic brain injury (TBI) and that pyruvate treatment acutely after TBI can improve cerebral metabolism and is neuroprotective. Since extracellular levels of glucose decrease during periods of increased cognitive demand and exogenous glucose improves cognitive performance, we hypothesized that pyruvate treatment prior to testing could ameliorate cognitive deficits in rats with TBI. Based on pre-surgical spatial alternation performance in a 4-arm plus-maze, adult male rats were randomized to receive either sham injury or unilateral (left) cortical contusion injury (CCI). On days 4, 9 and 14 after surgery animals received an intraperitoneal injection of either vehicle (Sham-Veh, n = 6; CCI-Veh, n = 7) or 1000 mg/kg of sodium pyruvate (CCI-SP, n = 7). One hour after each injection rats were retested for spatial alternation performance. Animals in the CCI-SP group showed no significant working memory deficits in the spatial alternation task compared to Sham-Veh controls. The percent four/five alternation scores for CCI-Veh rats were significantly decreased from Sham-Veh scores on days 4 and 9 (p < 0.01) and from CCI-SP scores on days 4, 9 and 14 (p < 0.05). Measures of cortical contusion volume, regional cerebral metabolic rates of glucose and regional cytochrome oxidase activity at day 15 post-injury did not differ between CCI-SP and CCI-Veh groups. These results show that spatial alternation testing can reliably detect temporal deficits and recovery of working memory after TBI and that delayed pyruvate treatment can ameliorate TBI-induced cognitive impairments.