Caspase-independent Cell Killing by Fas-associated Protein with Death Domain

The binding of Fas ligand to Fas recruits caspase 8 to Fas via an adaptor, FADD/MORT1, and activates a caspase cascade leading to apoptosis. Here, we describe a human Jurkat-derived cell line (JB-6) that is deficient in caspase 8. This cell line was resistant to the apoptosis triggered by Fas engagement. However, the multimerization of Fas-associated protein with death domain, through the use of a dimerizing system, killed the JB-6 cells. This killing process was not accompanied by the activation of caspases or DNA fragmentation. The dying cells showed neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swelled in a manner similar to that seen in necrosis. These results suggested that Fas-associated protein with death domain can kill the cells via two pathways, one mediated by caspases and another that does not involve them.     

Fanconi Anemia C Protein Acts at a Switch between Apoptosis and Necrosis in Mitomycin C-Induced Cell Death

Deregulation of apoptosis seems to be a hallmark of the Fanconi anemia (FA) syndrome. In order to further define the role of the FA protein from complementation group C (FAC) in apoptosis, we characterized parameters modified during the mitomycin-C (MMC)-induced apoptotic program. It is shown that despite a higher level of cell death for FA compared to normal lymphoblasts after MMC treatment, FA cells do not display a marked DNA fragmentation. Furthermore, while playing a central role in MMC apoptosis of normal lymphoblasts, the activity of caspase-3-like proteases is altered in FA cells. Interestingly, the disruption of the mitochondrial transmembrane potential (Δψ), an early event that can lead to apoptotic or to necrotic death, is accomplished similarly in FA and in normal cells. Finally, it is shown that the overexpressed FAC protein inhibited the apoptotic steps, with the exception of the decrease of the Δψ. Altogether, our results indicate that the FAC protein acts at a step preceding the activation of the caspases and after the modification of the Δψ, a decision point at which cells can be pushed toward either apoptosis or necrosis and which, consequently, regulates the balance between the two modes of cell death.     

Apoptosis of Cortical Neurons in Ischemic Insult

Acute brain ischemia is accompanied by the intense apoptotic and/or necrotic death of cortical neurons. This review deals with the molecular mechanisms underlying apoptosis, in particular those activated in progressive cerebral ischemic insult. We analyze the data of experimental studies and clinical findings that confirm the principal role of caspase-dependent cell death resulting from acute disorder of the brain circulation. The prospects for the use of apoptosis inhibitors in neurological practice for prevention or minimization of cerebral ischemic injury and reduction of neuronal degeneration within a penumbral zone are discussed.     

Hippocampal Membranes Contain a Neurotrophic Activity That Stimulates Cholinergic Properties of Fetal Rat Septal Neurons Cultured Under Serum-Free Conditions

Primary cultures of fetal rat septal neurons were used to identify a membrane-associated cholinergic neurotrophic activity. Under serum-free culture conditions, approximately 98% of the septal cells are neurons, and approximately 6% of the neurons are cholinergic as determined immunocytochemically. Crude membranes prepared from rat hippocampal homogenates stimulate choline acetyltransferase (ChAT) activity in treated septal neurons. The membrane-associated trophic activity is apparent at lower protein concentrations than activity present in the soluble fraction and is unevenly distributed in various brain regions; it is highest in hippocampus and striatum and negligible in cerebellum. Membrane trophic activity is developmentally regulated, is heat and trypsin sensitive, and increases the rate of expression of ChAT in septal neurons. Upon gel filtration chromatography of a high-salt membrane extract, trophic activity elutes as a broad peak in the 500 kilodalton (kD) molecular mass range. Stimulation of septal neuronal ChAT activity by either crude membranes or partially purified preparations is not inhibited by antibodies against nerve growth factor (NGF), and its maximal activity is additive to maximally active doses of NGF. The results indicate that hippocampal membranes contain cholinergic neurotrophic activity which may be important for the development of septal cholinergic neurons.     

Role of Ionic Fluxes in the Apoptotic Cell Death of Cultured Cerebellar Granule Neurons

Cultured cerebellar granule neurons (CGC) increase survival in a medium containing 25 mM KCl (K25), and they die apoptotically when cultures are treated with staurosporine (St) or are transferred to a 5-mM KCl containing medium (K5). Apoptotic CGC show nuclear condensation and caspase-3 activation. Cell death induced by these conditions was partially prevented when cultures were maintained under alkaline conditions, which also induced a marked reduction of the caspase-3 activation. The acidification of the medium further increased cell death induced by both stimuli. Cultures transferred to K5 suffered an immediate intracellular alkalinization that remained constant during the time K5 was present. In contrast, St did not modify cytosolic pH at any of the evaluated times. On the other hand, DIDS, furosemide, and bumetanide prevented CGC death induced by K5 and St. Other drugs such as amiloride, EIPA, tamoxifen, NEM, or NPPB did not modify cell death induced by these conditions. Both DIDS and bumetanide markedly inhibited the processing and activation of caspase-3, and DIDS prevented the nuclear condensation induced by K5 and St. These findings suggest that pH is a condition that could contribute to the modulation of cell death induced by some stimuli and that other ions, such as potassium, could have a role in the initial phase of apoptotic death of CGC.     

Flow cytometric scoring of apoptosis compared to electron microscopy in gamma irradiated lymphocytes

One of the early events occurring at the cell membrane during apoptosis is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer. These phosphatidylserine groups can be bound by fluorescein isothiocyanate (FITC)-labelled annexin V. The aim of this study was to evaluate the power of the annexin V flow cytometric assay in detecting apoptosis in gamma irradiated peripheral blood lymphocytes and in differentiating between apoptosis and primary necrosis in these cells. Therefore, 5 Gy and 20 Gy gamma irradiated peripheral blood mononuclear cells (PBMCs) were examined after a 24-h culture period. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique was performed as well. A comparison with an electron microscopic (EM) evaluation was made. EM is based on established morphological criteria allowing the classification of cells into four groups: viable, early apoptotic, secondary necrotic and primary necrotic cells. EM performed on annexin V positive sorted cells proved that a 5 Gy gamma irradiation of PBMCs mainly causes apoptosis, whereas a 20 Gy gamma irradiation mainly induces primary necrosis. Neither the annexin V flow cytometric assay nor the TUNEL assay were able to distinguish between primary and secondary necrotic cells. These results illustrate that if quantification of apoptosis is required, one should be careful in interpreting flow cytometric results obtained by annexin V or TUNEL staining in peripheral blood lymphocytes. Although in general primary necrotic cells show an increased forward scatter due to cellullar swelling, both early apoptotic and necrotic (primary or secondary) lymphocytes show a decreased forward scatter signal. Moreover, both primary and secondary necrotic lymphocytes are annexin V and propidium iodide (PI) positive and therefore indistinguishable. We conclude that if a new experiment focusing on apoptosis is set up, an initial EM evaluation is mandatory. If EM shows that the apoptosis inducing agent used in the design of the experiments is not causing primary necrosis, than the annexin V flow cytometric assay can provide rapid and quantitative information about apoptosis.     

Characterization of Na+-Dependent Phosphate Uptake in Cultured Fetal Rat Cortical Neurons

Abstract: Our laboratory has recently cloned and expressed a brain- and neuron-specific Na⁺-dependent inorganic phosphate (P_i) cotransporter that is constitutively expressed in neurons of the rat cerebral cortex, hippocampus, and cerebellum. We have now characterized Na⁺-dependent ³²P_i cotransport in cultured fetal rat cortical neurons, where >90% of saturable P_i uptake is Na⁺-dependent. Saturable, Na⁺-dependent ³²P_i uptake was first observed in primary cultures of cortical neurons at 7 days in vitro (DIV) and was maximal at 12 DIV. Na⁺-dependent P_i transport was optimal at physiological temperature (37°C) and pH (7.0–7.5), with apparent K_m values for P_i and Na⁺ of 54 ± 12.7 µM and 35 ± 4.2 mM, respectively. A reduction in extracellular Ca²⁺ markedly reduced (>60%) Na⁺-dependent P_i uptake, with a threshold for maximal P_i import of 1–2.5 mM CaCl₂. Primary cultures of fetal cortical neurons incubated in medium where equimolar concentrations of choline were substituted for Na⁺ had lower levels of ATP and ADP and higher levels of AMP than did those incubated in the presence of Na⁺. Furthermore, a substantial fraction of the ³²P_i cotransported with Na⁺ was concentrated in the adenine nucleotides. Inhibitors of oxidative metabolism, such as rotenone, oligomycin, or dinitrophenol, dramatically decreased Na⁺-dependent P_i import rates. These data establish the presence of a Na⁺-dependent P_i cotransport system in neurons of the CNS, demonstrate the Ca²⁺-dependent nature of ³²P_i uptake, and suggest that the neuronal Na⁺-dependent P_i cotransporter may import P_i required for the production of high-energy compounds vital to neuronal metabolism.     

An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as “apoptosis-necrosis continuum.” To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.     

Effect of Hyperoxia on Cortical Neuronal Nuclear Function and Programmed Cell Death Mechanisms

There is growing concern over detrimental neurologic effects to human newborns caused by increased inspired oxygen concentrations. We hypothesize that hyperoxia (FiO₂ > 0.95) results in increased high-affinity Ca²⁺-ATPase activity, Ca²⁺-influx, and proapoptotic protein expression in cortical neuronal nuclei of newborn piglets. Neuronal cerebral energy metabolism was documented by determining ATP and phosphocreatine levels. Neuronal nuclear conjugated dienes and fluorescent compounds were measured as indices of lipid peroxidation. High-affinity Ca²⁺-ATPase activity and ATP-dependent Ca²⁺-influx were determined to document neuronal nuclear membrane function. Hyperoxia resulted in increases in lipid peroxidation, high-affinity Ca²⁺-ATPase activity, ATP-dependent Ca²⁺-influx, and Bax/Bcl-2 ratio in the cortical neuronal nuclei of newborn piglets. We conclude that hyperoxia results in modification of neuronal nuclear membrane function leading to increased nuclear Ca²⁺-influx, and propose that hyperoxia-induced increases in intranuclear Ca²⁺ activates the Ca²⁺/calmodulin-dependent protein kinase pathway, triggering increased CREB protein-mediated apoptotic protein expression in hyperoxic neurons.     

Role of the Outward Delayed Rectifier K+ Current in Ceramide-Induced Caspase Activation and Apoptosis in Cultured Cortical Neurons

We studied the novel hypothesis that an up-modulation of channels for outward delayed rectifier K+ current (I(K)) plays a key role in ceramide-induced neuronal apoptosis. Exposure for 6-10 h to the membrane-permeable C2-ceramide (25 microM) or to sphingomyelinase (0.2 unit/ml), but not to the inactive ceramide analogue C2-dihydroceramide (25 microM), enhanced the whole-cell I(K) current without affecting the transient A-type K+ current and increased caspase activity, followed by neuronal apoptosis 24 h after exposure onset. Tetraethylammonium (TEA) or 4-chloro-N,N-diethyl-N-heptylbenzenebutanaminium tosylate (clofilium), at concentrations inhibiting I(K), attenuated the C2-ceramide-induced caspase-3-like activation as well as neuronal apoptosis. Raising extracellular K+ to 25 mM similarly blocked the C2-ceramide-induced cell death; the neuroprotection by 25 mM K+ or TEA was not eliminated by blocking voltage-gated Ca2+ channels. An inhibitor of tyrosine kinases, herbimycin A (10 nM) or lavendustin A (0.1-1 microM), suppressed I(K) enhancement and/or apoptosis induced by C2-ceramide. It is suggested that ceramide-induced I(K) current enhancement is mediated by tyrosine phosphorylation and plays a critical role in neuronal apoptosis.     

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability^1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells³. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells ^4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane ^1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases^7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence ^1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment¹⁰. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence¹⁰. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).     

动物源杂合肽P18对白血病K562细胞的致死作用研究

通过其致死白血病K562细胞过程,对动物源杂合肽P18的分子结构与功能进行了研究.MTT实验表明P18显著致死K562细胞;Western blot实验和流式细胞分析提示P18导致 K562细胞坏死,而非依赖于caspases的传统凋亡;荧光探针标记后,观察到P18显著增加K562细胞的膜通透性;圆二(CD)图谱分析,在模拟细胞膜环境的溶液中,P18分子出现α-螺旋.综上所述,P18肽分子可能在细胞膜微环境的介导下产生α-螺旋,产生或暴露了肽分子的破膜结构,导致膜通透性显著增加,最终导致K562细胞的坏死.     

Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death

Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl₂ and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.     

Measurement of apoptosis in cytological specimens by flow cytometry: comparison of Annexin V,caspase cleavage and dUTP incorporation assays

H. P. Dong, A. Holth, M. G. Ruud, E. Emilsen, B. Risberg and B. Davidson Measurement of apoptosis in cytological specimens by flow cytometry: comparison of annexin V, caspase cleavage and dUTP incorporation assays Objective: To compare the performance of different assays for measuring apoptosis in cytological specimens. Methods: Apoptosis was assessed in 27 specimens (22 effusions, five fine needle aspirates; 20 malignant, seven reactive) using flow cytometry, applying assays for the measurement of annexin V expression, caspase‐3 and ‐8 cleavage and deoxynucleotidyl transferase deoxyuridine triphosphates (dUTP) incorporation. Results were studied for differences between reactive and malignant specimens, as well as performance across assays. Results: Wide variation in the degree of apoptosis was observed in both benign and malignant specimens using all assays. However, the percentage of annexin V‐positive cells was higher compared with those showing caspase cleavage or dUTP incorporation in the majority of cases, irrespective of specimen type. Comparative analysis of benign and malignant specimens showed no significant differences in expression of any of the studied parameters. However, tumour cells and reactive mesothelial cells in pleural effusions had a significantly lower level of dUTP incorporation compared with their counterparts in peritoneal specimens (P = 0.001). Conclusions: The present data are in agreement with our previous observation in ovarian carcinoma effusions, that measurement of apoptosis by the annexin V assay provides higher expression values than those obtained by other assays, suggesting that this assay does not accurately reflect the degree of apoptosis in benign or malignant cells in effusions.     

Psychosine Is as Potent an Inducer of Cell Death as C6-Ceramide in Cultured Fibroblasts and in MOCH-1 Cells

Cytotoxic capacity of psychosine (galactosylsphingosine) was evaluated in comparison with C6-ceramide in cultured fibroblasts and the glia-derived MOCH-1 cells that have characteristics of myelinating cells (1). Psychosine caused cytotoxic cell death and DNA fragmentation at concentrations similar to C6-ceramide and MOCH-1 cells were substantially more sensitive to their cytotoxic effects than fibroblasts. In this system, pretreatment with GM1-ganglioside failed to protect the cells from the deleterious effects of these compounds. These findings are consistent with the hypothesis that psychosine is the cytotoxic metabolite that causes apoptotic death of the oligodendrocyte in globoid cell leukodystrophy (Krabbe disease). They further suggest that the protective capacity of GM1-ganglioside is unlikely to be the explanation for the paradoxical improvement of the phenotype of globoid cell leukodystrophy in the mouse simultaneously deficient in two lysosomal -galactosidases, galactosylceramidase and GM1-ganglioside -galactosidase.     

C02倍增对渗透胁迫下小麦叶片抗氧化酶类及细胞程序性死亡的影响

经渗透胁迫后 ,CO2 倍增条件下小麦叶片的SOD、POX和CAT的活性均显著高于对照 ,上升或稳定时期较长 ;在渗透胁迫后期MDA含量和电解质泄露率增加较慢 ,显著低于对照 ;H2 O2 含量一直高于对照但进行PEG胁迫后增长较慢。CO2 倍增条件下 ,小麦细胞出现DNA梯的时间较晚而且持续的时间较长 ,DNA梯出现时抗氧化酶和H2 O2 处于相对稳定状态。结果表明在渗透胁迫下CO2 倍增使小麦的抗氧化能力增强从而减轻了对细胞膜和DNA的损伤 ,并且干旱条件下小麦的细胞程序性死亡可能是由于细胞内氧化过强所致     

The spread of apoptosis through gap-junctional channels in BHK cells transfected with Cx32

Programmed cell death (apoptosis) occurs both during normal development and as a result of various pathological conditions. An in vitro system was used to explore the transmission of death signals from apoptotic cells to cells with which they were coupled via gap junctions. Confluent cultures of baby hamster kidney (BHK) cells, stably transfected with the gap-junctional protein connexin32, were scrape loaded with cytochrome C (cyC), a mitochondria-derived apoptotic agent, to introduce the protein into cells injured by the cut. The cultures were subsequently analyzed for the presence of activated caspases, the distribution of TUNEL staining, and the binding of annexin V. Although cyC is too large to traverse the gap junctional channel, each of the assays revealed that apoptosis had spread from dying cells at the margin of the scrape to otherwise healthy neighboring cells to which they were coupled. This ‘bystander effect’ was significantly reduced in the presence of agents that block gap junctional intercellular communication.     

Non-apoptogenic killing of hela cervical carcinoma cells after short exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

We examined the action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on HeLa cells and compared it with that of cisplatin (CP). MNNG directly killed a substantial number of cells within 1 hour and resulted in strong DNA-damage as evidenced by Comet measurements. Despite appearance of DNA lesions, p53 protein was not activated. Analysis of HeLa cells treated with MNNG for 1h, 3h and 6h by flow cytometry and by Hoechst staining did not reveal any sub-G(1) cell population and chromatin condensation/fragmentation characteristic for apoptosis, respectively. Also, no biochemical changes typical for apoptosis such as activation of caspase-3 or release of cytochrome C from mitochondria were detected. Inactivation of PARP-1 reduced the direct cytotoxicity exerted by MNNG. Our results showing that despite appearance of severe DNA lesions after short exposure of HeLa cells to MNNG neither activation of p53 response nor induction of apoptosis occurred implicate that generation of strong DNA damage is not sufficient to stabilize p53 protein in HeLa cells. Our data unequivocally show that the conscientious determination of the type of cell death induced by genotoxic agents is necessary. The assessment of the changes based on at least a few independent criteria is required to discriminate between apoptosis and necrosis. Since the alkylating agents generate DNA strand breaks, the recruitment of methods based on determination of DNA cleavage such as DNA ladder or TUNEL assay for evaluation of apoptosis is not adequate.     

Mitochondrial Events in the Life and Death of Animal Cells: A Brief Overview

Traditionally, mitochondria have been viewed as the powerhouse of the cell, i.e., the site of theoxidative phosphorylation machinery involved in ATP production. Consequently, much of theresearch conducted on mitochondria over the past 4 decades has focused on elucidating both thosemolecular events involved in ATP synthesis by oxidative phosphorylation and those involved inthe biogenesis of the oxidative phosphorylation machinery. While monumental achievements havebeen made, and continue to be made, in the study of these remarkable but extremely complexprocesses essential for the life of most animal cells, it has been only in recent years that a largebody of biological and biomedical scientists have come to recognize that mitochondria participatein other important processes. Two of these are cell death and aging which, not surprisingly, are relatedprocesses both involving, in part, the oxidative phosphorylation machinery. This new awareness hassparked a new and growing area of mitochondrial research, that has become of great interest to awide variety of scientists ranging from those involved in elucidating the role of mitochondria incell death and aging to those interested in either suppressing or facilitating these processes as itrelates to identifying new therapies or drugs for human disease. It is the purpose of this briefintroductory review to provide an overview of those mitochondrial events involved in the life anddeath of animal cells and to indicate how these events might relate to the human aging process.Much more is known, much remains controversial, and even more remains to be learned as indicatedin the excellent set of minireviews that follow.     

Cell death in the injured brain: Roles of metallothioneins

In traumatic brain injury (TBI), the primary, irreversible damage associated with the moment of impact consists of cells dying from necrosis. This contributes to fuelling a chronic central nervous system (CNS) inflammation with increased formation of proinflammatory cytokines, enzymes and reactive oxygen species (ROS). ROS promote oxidative stress, which leads to neurodegeneration and ultimately results in programmed cell death (secondary injury). Since this delayed, secondary tissue loss occurs days to months following the primary injury it provides a therapeutic window where potential neuroprotective treatment could alleviate ongoing neurodegeneration, cell death and neurological impairment following TBI. Various neuroprotective drug candidates have been described, tested and proven effective in pre-clinical studies, including glutamate receptor antagonists, calcium-channel blockers, and caspase inhibitors. However, most of the scientific efforts have failed in translating the experimental results into clinical trials. Despite intensive research, effective neuroprotective therapies are lacking in the clinic, and TBI continues to be a major cause of morbidity and mortality.This paper provides an overview of the TBI pathophysiology leading to cell death and neurological impairment. We also discuss endogenously expressed neuroprotectants and drug candidates, which at this stage may still hold the potential for treating brain injured patients.