Dye-coupling between glial cells in the guinea pig neocortical slice

Physiologically identified glial cells in guinea pig neocortical slices were injected with the low molecular weight, fluorescent dye Lucifer yellow CH. The stained aggregates which resulted consisted of one brightly stained, central cell surrounded by numerous lightly stained cells. The central cell had well defined feathery processes and resembled a protoplasmic astrocyte. The surrounding cells appeared also to be glial cells but lacked sufficient detail to be further categorized. This first demonstration of dye-coupling between neocortical glial cells strongly suggests that these cells are connected together via low resistance junctions capable of passing ionic current as well as dye.     

Neurogenesis and glial proliferation are stimulated following diffuse traumatic brain injury in adult rats

Although increased neurogenesis has been described in rodent models of focal traumatic brain injury (TBI), the neurogenic response occurring after diffuse TBI uncomplicated by focal injury has not been examined to date, despite the pervasiveness of this distinct type of brain injury in the TBI patient population. Here we characterize multiple stages of neurogenesis following a traumatic axonal injury (TAI) model of diffuse TBI as well as the proliferative response of glial cells. TAI was induced in adult rats using an impact-acceleration model, and 5-bromo-2'-deoxyuridine (BrdU) was administered on days 1-4 posttrauma or sham operation to label mitotic cells. Using immunohistochemistry for BrdU combined with phenotype-specific markers, we found that proliferation was increased following TAI in the subventricular zone of the lateral ventricles and in the hippocampal subgranular zone, although the ultimate production of new dentate granule neurons at 8 weeks was not significantly enhanced. Also, abundant proliferating and reactive astrocytes, microglia, and polydendrocytes were detected throughout the brain following TAI, indicating that a robust glial response occurs in this model, although very few new cells in the nonneurogenic brain regions became mature neurons. We conclude that diffuse brain injury stimulates early stages of a neurogenic response similar to that described for models of focal TBI.     

Brain remodeling due to neuronal and astrocytic proliferation after controlled cortical injury in mice.

The persistence of neural stem cells into adulthood has been an area of intense investigation in recent years. There is limited knowledge about how an acquired brain injury might affect the ability of neural precursor cells to proliferate and repopulate injured areas. In the present study we utilize a controlled cortical impact model of traumatic brain injury in adult mice and subsequent BrdU labeling to demonstrate that there is significant proliferation of neural precursors in response to traumatic brain injury in areas both proximal and distal to the injury site. The fate of the proximal proliferation is almost exclusively astrocytic at 60-days post injury and demonstrates that newly generated cells make up much of the astrogliotic scar. Moreover, in areas more distal from the injury site, neurogenesis occurs within the granular layer of the dentate gyrus at a level more than five-fold greater than in controls. These data demonstrate that neural proliferation plays key roles in the remodeling that occurs after traumatic brain injury and suggests a mechanism as to how functional recovery after traumatic brain injuries continues to occur long after the injury itself.     

No DCX-positive neurogenesis in the cerebral cortex of the adult primate

Whether endogenous neurogenesis occurs in the adult cortex remains controversial.An increasing number of reports suggest that doublecortin(DCX)-positive neurogenesis persists in the adult primate cortex,attracting enormous attention worldwide.In this study,different DCX antibodies were used together with NeuN antibodies in immunohistochemistry and western blot assays using adjacent cortical sections from adult monkeys.Antibody adsorption,antigen binding,primary antibody omission and antibody-free experiments were used to assess specificity of the signals.We found either strong fluorescent signals,medium-weak intensity signals in some cells,weak signals in a few perikarya or near complete lack of labeling in adjacent cortical sections incubated with the various DCX antibodies.The putative DCX-positive cells in the cortex were also positive for NeuN,a specific marker of mature neurons.However,further experiments showed that most of these signals were either the result of antibody cross reactivity,the non-specificity of secondary antibodies or tissue autofluorescence.No confirmed DCX-positive cells were detected in the adult macaque cortex by immunofluorescence.Our findings show that DCX-positive neurogenesis does not occur in the cerebral cortex of adult primates,and that false-positive signals(artefacts)are caused by antibody cross reactivity and autofluorescence.The experimental protocols were approved by the Institutional Animal Care and Use Committee of the Institute of Neuroscience,Beijing,China(approval No.IACUC-AMMS-2014-501).     

Endogenous adult neurogenesis and cognitive function recovery following traumatic brain injury in the rat hippocampus

BACKGROUND:Endogenous neural progenitor cells play a beneficial role for cognitive recovery following traumatic brain injury.However,there are few classification-control studies aimed at varying graded brain trauma.OBJECTIVE:To observe the effects of adult endogenous neurogenesis on cognitive function repair and regeneration of neural progenitor cells following varying graded traumatic hippocampal injury to determine the significance of endogenous neurogenesis in the repair of brain injury.DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Key Laboratory of Injuries,Variations and Regeneration of Nervous System,Tianjin Medical University General Hospital,from February to October 2009.MATERIALS:Mouse anti-rat 5-bromodeoxyuridine (BrdU) and neuronal nuclei (NeuN) monoclonal antibodies were purchased from Millipore Corporation,USA.METHODS:A total of 45 Wistar rats were randomly assigned to three groups.Mild and severe injury groups were respectively subjected to (182 ± 2) kPa and (284 ± 4) kPa lateral fluid percussion to establish models of brain injury,and the control group was subjected to surgery with no lateral fluid percussion.MAIN OUTCOME MEASURES:Cognitive function was estimated using the Morris water maze.Proliferation,survival,and differentiation of newly generated cells in the injured hippocampus were observed through the use of immunofluorescent staining.RESULTS:At 7 days post-injury,the number of BrdU+ cells in the hippocampal dentate gyrus significantly increased in the mild and severe injury groups compared with the control group (P<0.01).At 61 days post-injury,the number of BrdU7NeuN+ cells in the hippocampal dentate gyrus was significantly greater in the mild injury group compared with the severe injury and control groups (P< 0.01).In addition,the control group exhibited the greatest proportion of surviving cells that differentiated into mature neurons compared with the injury groups (P< 0.01).Moreover,at 61 days post-injury,cognitive function in rats with mild injury recovered to normal levels,whereas the severe injury group exhibited cognitive deficits (P< 0.01).CONCLUSION:Traumatic brain injury may be a stimulation factor for proliferation of neural progenitor cells in the adult hippocampus but severe brain trauma does not lead to an increased number of newly generated cells.Endogenous adult neurogenesis repairs neurological functions to an extent.However,recovery of neurological function remains limited following severe traumatic brain injury.     

Distinct effect of impact rise times on immediate and early neuropathology after brain injury in juvenile rats

Traumatic brain injury (TBI) can occur from physical trauma from a wide spectrum of insults ranging from explosions to falls. The biomechanics of the trauma can vary in key features, including the rate and magnitude of the insult. Although the effect of peak injury pressure on neurological outcome has been examined in the fluid percussion injury (FPI) model, it is unknown whether differences in rate of rise of the injury waveform modify cellular and physiological changes after TBI. Using a programmable FPI device, we examined juvenile rats subjected to a constant peak pressure at two rates of injury: a standard FPI rate of rise and a faster rate of rise to the same peak pressure. Immediate postinjury assessment identified fewer seizures and relatively brief loss of consciousness after fast‐rise injuries than after standard‐rise injuries at similar peak pressures. Compared with rats injured at standard rise, fewer silver‐stained injured neuronal profiles and degenerating hilar neurons were observed 4–6 hr after fast‐rise FPI. However, 1 week postinjury, both fast‐ and standard‐rise FPI resulted in hilar cell loss and enhanced perforant path‐evoked granule cell field excitability compared with sham controls. Notably, the extent of neuronal loss and increase in dentate excitability were not different between rats injured at fast and standard rates of rise to peak pressure. Our data indicate that reduced cellular damage and improved immediate neurological outcome after fast rising primary concussive injuries mask the severity of the subsequent cellular and neurophysiological pathology and may be unreliable as a predictor of prognosis. © 2014 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.     

Endogenous versus exogenous markers of adult neurogenesis in canaries and other birds: Advantages and disadvantages

Although the existence of newborn neurons had originally been suggested, but not broadly accepted, based on studies in adult rodent brains, the presence of an active neurogenesis process in adult homoeothermic vertebrates was first firmly established in songbirds. Adult neurogenesis was initially studied with the tritiated thymidine technique, later replaced by the injection and detection of the marker of DNA replication 5‐bromo‐2′‐deoxyuridine (BrdU). More recently, various endogenous markers were used to identify young neurons or cycling neuronal progenitors. We review here the respective advantages and pitfalls of these different approaches in birds, with specific reference to the microtubule‐associated protein, doublecortin (DCX), that has been extensively used to identify young newly born neurons in adult brains. All these techniques of course have limitations. Exogenous markers label cells replicating their DNA only during a brief period and it is difficult to select injection doses that would exhaustively label all these cells without inducing DNA damage that will also result in some form of labeling during repair. On the other hand, specificity of endogenous markers is difficult to establish due to problems related to the specificity of antibodies (these problems can be, but are not always, addressed) and more importantly because it is difficult, if not impossible, to prove that a given marker exhaustively and specifically labels a given cell population. Despite these potential limitations, these endogenous markers and DCX staining in particular clearly represent a useful approach to the detailed study of neurogenesis especially when combined with other techniques such as BrdU. J. Comp. Neurol. 522:4100–4120, 2014. © 2014 Wiley Periodicals, Inc.     

急性硬脑膜下血肿减压术后对侧进展性硬脑膜外血肿的早期诊治

目的 探讨颅脑外伤去骨瓣减压术(DC)后对侧进展性硬脑膜外血肿(EDH)的临床特点和早期诊治方法.方法 对23例颅脑外伤术后对侧进展性EDH患者的资料进行回顾性分析.结果 术后对侧进展性EDH的平均确诊时间为(4±3)h;EDH的部位为颞顶部12例,枕顶部9例,额颞部2例;主要表现为术中脑肿胀8例,术后对侧瞳孔散大4例,术后ICP逐渐增高4例,神经系统无明显异常改变者7例.保守治疗1例,再手术22例,21例确认血肿处骨折存在.出院时GOS评分5分5例,4分7例,3分7例,2分者1例,1分3例.结论 急性外伤性硬脑膜下血肿(SDH)术后对侧进展性EDH以颞顶和枕顶部最多见,冲击部位颅骨骨折处出血是最主要的机制,早期确诊和治疗有助于改善预后.     

重型颅脑损伤并发脑梗死的危险因素分析

目的探讨重型颅脑损伤并发脑梗死的危险因素,为临床诊治提供参考。方法回顾性分析2015年1月至2016年12月收治的符合标准的172例重型颅脑损伤的临床资料,采用多因素Logistic回归分析检验危险因素。结果 172例中,44例并发脑梗死,128例无脑梗死。多因素Logistic回归分析表明脑疝、弥漫性脑肿胀、失血性休克是重型颅脑损伤并发脑梗死的独立危险因素。结论对于伴有脑疝、弥漫性脑肿胀、失血性休克的成人重型颅脑损伤,临床应注意防止脑梗死。     

颅脑对冲伤与冲击伤再手术的预见性分析

目的探讨颅脑对冲伤与冲击伤再手术的原因和预见性因素。方法回顾性分析18例颅脑对冲伤与冲击伤再手术患者的临床特点。结果本组18例采用GOS评定,随访6个月～2年,良好5例,轻残5例,重残2例,植物生存1例,死亡5例。结论,颅脑外伤同时具备减速性损伤,冲击伤侧的颅骨骨折,时间短,对冲伤侧的硬膜下血肿宽度大于10cm,意识昏迷时再手术可能性大。     

急性颅脑损伤后迟发性血肿的诊治经验

目的探讨如何及时发现急性颅脑损伤后迟发性血肿及治疗。方法对我院38例迟发性外伤性颅内血肿均行手术治疗病人的临床资料进行回顾性分析和总结。结果出院时患者G0S分级:V级14例,Ⅳ级10例,Ⅲ级6例,Ⅱ级2例,Ⅰ级6例。预后优良率(良好+中残)为63.2%(24/38),病死率为15.8%(6/38)。结论迟发性血肿形成有可预见性因素和明显临床特点,对有高危因素者,应加强观察和监护,宜动态复查CT,早诊断早治疗以提高疗效。     

Regional and temporal characterization of neuronal, glial, and axonal response after traumatic brain injury in the mouse

Fluid percussion injury (FPI) is a commonly used and clinically relevant model of traumatic brain injury (TBI) in the rat. Recently, our lab successfully adapted FPI to mice. To account for differences in response to injury between mice and rats and provide a foundation for further use of FPI in gene-targeting studies, we sought to characterize the temporal and regional response to FPI in male C57BL/6 mice. Animals were killed at 10 min, 24 h, and 4, 7, 14, and 35 days (n = 3 for each group) after a very severe parasagittal FPI (> 4.0 atm) or sham injury (n = 3). Extensive numbers of damaged neurons were consistently found in the ipsilateral cortex, thalamus, and hippocampus by 10 min. This damage was nearly identical at 24 h, but quickly declined at subsequent time points. Activated microglia were found only in regions of neuronal injury at the earliest time points. Glial fibrillary acidic protein immunoreactivity reached significantly higher levels compared with controls at 7 days (P < 0.05) in the cortex, thalamus, and hippocampus and remained elevated for 35 days. White matter degeneration was present in all regions examined. This damage did not appear until at least day 4, but progressed up to day 35. The spatial pattern of damage we observed in mice after FPI is similar to that seen in rats. However, the temporal progression of neuronal injury in mice is comparatively abbreviated in the hippocampus and thalamus. In conclusion, these results suggest that FPI in mice may be a particularly useful tool for studying mechanisms of TBI in gene-targeting studies. Received: 13 October 1998 / Revised: 22 February 1999 / Accepted: 24 March 1999     

颅脑创伤后脑梗死的诊断和治疗(附32例报道)

目的总结颅脑创伤后脑梗死的诊治经验。方法对收治的32例颅脑创伤后脑梗死患者的临床资料、影像学检查、治疗及结果进行回顾性总结。结果本组32例患者占同期收治的1 439例颅脑创伤患者的2.2%,车祸伤24例,坠落伤6例,打击伤2例。伤后有脑疝表现者21例,合并休克者12例,胸腹部伤者12例,多器官伤者10例。伤后首次头颅CT扫描均见占位性颅内血肿,其中中线结构偏移>10 mm者20例。本组脑梗死均经再次头颅CT扫描确诊,受伤至脑梗死确诊的平均时间为(24±12)h;临床上表现为术中脑肿胀18例,术后意识状态无好转14例。再手术减压3例。出院时死亡20例,植物状态生存7例,重残3例,中残2例。结论颅脑创伤后大脑半球梗死的预后很差,脑疝、失血性休克、合并多发伤是颅脑创伤后大脑半球梗死的危险因素,早期诊断和及时处置伤后颅内高压是改善其预后的关键。     

中重度颅脑损伤继发急性脑梗死的相关危险因素和预后分析

目的探讨中重度颅脑外伤患者继发急性外伤性脑梗死(PTCI)的危险因素和预后。方法回顾性分析218例中重度颅脑外伤患者的临床资料,其中27例伤后1周内出现PTCI。通过多因素Logistic回归分析,分析患者的性别、年龄、入院格拉斯哥昏迷量表(GCS)评分、是否合并脑疝、颅内压、血压、血气分析中CO2值、DIC评分、是否合并感染、有无实施去骨瓣减压术共10个因素与PTCI的关系,以及对患者预后的影响。结果经过统计学分析,患者的入院GCS评分、合并脑疝、颅高压、低血压、DIC评分是发生PTCI的独立危险因素,而年龄、性别、CO2值、合并感染以及有无实施去骨瓣减压术与PTCI无明显相关性。入院GCS评分低(3~5分)、合并脑疝、低血压、DIC评分≥5分、合并感染、继发PTCI是患者预后不良的独立预测因素。结论中重度颅脑外伤患者入院GCS评分、合并脑疝、颅高压、低血压、DIC评分是发生PTCI的独立危险因素;入院GCS评分低、合并脑疝、低血压、DIC评分≥5分、合并感染、继发PTCI的患者预后差,致残率高。     

In vitro neuronal and glial differentiation from embryonic or adult neural precursor cells are differently affected by chronic or acute activation of microglia

The contribution of microglia to the modulation of neurogenesis under pathological conditions is unclear. Both pro- and anti-neurogenic effects have been reported, likely reflecting the complexity of microglial activation process. We previously demonstrated that prolonged (72 hr) in vitro exposure to lipopolysaccharide (LPS) endows microglia with a potentially neuroprotective phenotype, here referred as to "chronic". In the present study we further characterized the chronic phenotype and investigated whether it might differently regulate the properties of embryonic and adult neural precursor cells (NPC) with respect to the "acute" phenotype acquired following a single (24 hr) LPS stimulation. We show that the LPS-dependent induction of the proinflammatory cytokines interleukin (IL)-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha was strongly reduced after chronic stimulation of microglia, as compared with acute stimulation. Conversely, the synthesis of the anti-inflammatory cytokine IL-10 and the immunomodulatory prostaglandin E2 (PGE2) was still elevated or further increased, after chronic LPS exposure, as revealed by real time PCR and ELISA techniques. Acutely activated microglia, or their conditioned medium, reduced NPC survival, prevented neuronal differentiation and strongly increased glial differentiation, likely through the release of proinflammatory cytokines, whereas chronically activated microglia were permissive to neuronal differentiation and cell survival, and still supported glial differentiation. Our data suggest that, in a chronically altered environment, persistently activated microglia can display protective functions that favor rather than hinder brain repair processes.     

Downregulation of UBE2Q1 is associated with neuronal apoptosis in rat brain cortex following traumatic brain injury

Aberrant functionality of the ubiquitin proteasome system (UPS) has been implicated in the pathology of various neurological disorders. Although it has been reported that the expressions of various UPS components were altered significantly following traumatic brain injury (TBI), detailed information on the subject remains largely unclear. In the study, using microarray assay, we identified a gene encoding ubiquitin‐conjugating enzyme E2Q1 (UBE2Q1) that was significantly downregulated during TBI. Western blot and immunohistochemical analyses verified the reduced expression of UBE2Q1 in ipsilateral brain cortex adjacent to the lesion site compared with the contralateral and sham‐operated ones. Double‐immunofluorescence staining indicated that UBE2Q1 was expressed mainly in the nucleus of neurons, with a minority in astrocytes in normal cortex. In addition, we observed a remarkable reduction in the number of UBE2Q1‐positive neurons following brain trauma. Furthermore, we showed that TBI resulted in a significant increase in the levels of p53, bax, p21 and active caspase 3 in brain cortex, which was correlated with decreased expression of UBE2Q1. We also found that knockdown of UBE2Q1 apparently increased the level of p53, whereas overexpressing UBE2Q1 attenuated cellular p53 level in PC12 neuronal cells. Accordingly, interference with UBE2Q1 augmented H₂O₂‐induced apoptosis of PC12 cells. Taken together, our findings indicate that UBE2Q1 might play an important role in the neuropathological process of TBI through modulating p53 signaling. © 2013 Wiley Periodicals, Inc.     

Acupuncture promotes nerve repair through the benign regulation of mTOR‐mediated neuronal autophagy in traumatic brain injury rats

AimsRecent investigations have already proved the neuroprotective efficacy of acupuncture in clinical practice in the treatment of neurological diseases, such as traumatic brain injury (TBI). Since growing evidence has suggested that neuronal autophagy was involved in multiple stages of TBI, this study aims to clarify the autophagy mediating mechanism underlying the neuroprotective effect of acupuncture in TBI rats.MethodsThree experiments were carried out to detect changes in neuronal autophagy and identify the potential molecular mechanism underlying the neuroprotective effect of acupuncture for TBI treatment. Feeney''s free‐falling epidural impingement method was used to establish the moderate TBI rat model; modified neurological severity scoring (mNSS) was used for neurological recovery evaluation. Nissl and HE staining were used to examine the histopathological changes. Immunofluorescence was used to detect the LC3‐positive cell rate. The transmission electron microscope (TEM) was used to investigate the morphology and quantity of autophagosomes. Western blotting was used to determine the protein expressions of LC3, p62, beclin1, mTOR, ULK1, p‐mTOR, and p‐ULK1. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was used for gene expressions analysis of LC3 mRNA and p62 mRNA. Co‐immunoprecipitation (CO‐IP) method was used to identify the protein interaction of mTOR and ULK1.ResultsOn Day 3 after TBI, acupuncture accelerated the removal of damaged cellular structures by promoting neuronal autophagy; on Day 7 and Day 14 after TBI, acupuncture inhibited neuronal autophagy, preventing excessive autophagy and thus alleviated nerve damage. In addition, the simultaneous treatment with 3‐MA or rapamycin at different stages after TBI attenuated the effect of acupuncture.ConclusionAcupuncture has a benign regulatory effect on neuronal autophagy in different stages of TBI, possibly through the mTOR/ULK1 pathway.     

Mechanical injury to neuronal/glial cultures in microplates: Role of NMDA receptors and pH in secondary neuronal cell death

In vitro models of traumatic injury are useful adjuncts to animal models for studying mechanisms of post-traumatic cell death. Here we describe a new in vitro model in which reproducible levels of injury are delivered by a punch device that produces 28 parallel cuts in individual wells of 96-well microplates. Cell loss is measured by LDH assay or quantitative fluorometric assay for ethidium homodimer staining. Glial cultures show cell death restricted to the initial injury site, whereas neuronal/glial cultures demonstrate substantial spread of cell loss over time. We used this model to examine the role of pH and NMDA receptors in delayed post-traumatic injury. NMDA receptor blockade by dizocilpine (MK-801) or treatment with antisense oligodeoxynucleotides directed against NMDAR1 was neuroprotective. Decreased cell death was observed under acidic conditions whereas increased extracellular pH was associated with increased, MK-801 sensitive cell loss. Advantages of our model include: reproducible trauma induction; rapid measurements of cell injury; and use of 96-well microplates which reduce time and cost. This model appears to be well-suited for the study of selected mechanisms of post-traumatic neuronal injury as well as for screening potential neuroprotective agents. J. Neurosci. Res. 51:748–758, 1998. © 1998 Wiley-Liss, Inc.