Transferase-mediated biotinylated-dUTP Nick-end Labeling Research Articles

Preconditioning Exercise in Rats Attenuates Early Brain Injury Resulting from Subarachnoid Hemorrhage by Reducing Oxidative Stress, Inflammation, and Neuronal Apoptosis.

Subarachnoid hemorrhage (SAH) is a catastrophic form of stroke responsible for significant morbidity and mortality. Oxidative stress, inflammation, and neuronal apoptosis are important in the pathogenesis of early brain injury (EBI) following SAH. Preconditioning exercise confers neuroprotective effects, mitigating EBI; however, the basis for such protection is unknown. We investigated the effects of preconditioning exercise on brain damage and sensorimotor function after SAH. Male rats were assigned to either a sham-operated (Sham) group, exercise (Ex) group, or no-exercise (No-Ex) group. After a 3-week exercise program, they underwent SAH by endovascular perforation. Consciousness level, neurological score, and sensorimotor function were studied. The expression of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), 4-hydroxynonenal (4HNE), nitrotyrosine (NT), ionized calcium-binding adaptor molecule 1 (Iba1), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β), 14-3-3γ, p-β-catenin Ser37, Bax, and caspase-3 were evaluated by immunohistochemistry or western blotting. The terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) assay was also performed. After SAH, the Ex group had significantly reduced neurological deficits, sensorimotor dysfunction, and consciousness disorder compared with the No-Ex group. Nrf2, HO-1, and 14-3-3γ were significantly higher in the Ex group, while 4HNE, NT, Iba1, TNF-α, IL-6, IL-1β, Bax, caspase-3, and TUNEL-positive cells were significantly lower. Our findings suggest that preconditioning exercise ameliorates EBI after SAH. The expression of 4HNE and NT was reduced by Nrf2/HO-1 pathway activation; additionally, both oxidative stress and inflammation were reduced. Furthermore, preconditioning exercise reduced apoptosis, likely via the 14-3-3γ/p-β-catenin Ser37/Bax/caspase-3 pathway.

B-Cell Lymphoma 2 (Bcl-2) and Regulation of Apoptosis after Traumatic Brain Injury: A Clinical Perspective.

Background and Objectives: The injury burden after head trauma is exacerbated by secondary sequelae, which leads to further neuronal loss. B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein and a key modulator of the programmed cell death (PCD) pathways. The current study evaluates the clinical evidence on Bcl-2 and neurological recovery in patients after traumatic brain injury (TBI). Materials and Methods: All studies in English were queried from the National Library of Medicine PubMed database using the following search terms: (B-cell lymphoma 2/Bcl-2/Bcl2) AND (brain injury/head injury/head trauma/traumatic brain injury) AND (human/patient/subject). There were 10 investigations conducted on Bcl-2 and apoptosis in TBI patients, of which 5 analyzed the pericontutional brain tissue obtained from surgical decompression, 4 studied Bcl-2 expression as a biomarker in the cerebrospinal fluid (CSF), and 1 was a prospective randomized trial. Results: Immunohistochemistry (IHC) in 94 adults with severe TBI showed upregulation of Bcl-2 in the pericontusional tissue. Bcl-2 was detected in 36–75% of TBI patients, while it was generally absent in the non-TBI controls, with Bcl-2 expression increased 2.9- to 17-fold in TBI patients. Terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick-end labeling (TUNEL) positivity for cell death was detected in 33–73% of TBI patients. CSF analysis in 113 TBI subjects (90 adults, 23 pediatric patients) showed upregulation of Bcl-2 that peaked on post-injury day 3 and subsequently declined after day 5. Increased Bcl-2 in the peritraumatic tissue, rising CSF Bcl-2 levels, and the variant allele of rs17759659 are associated with improved mortality and better outcomes on the Glasgow Outcome Score (GOS). Conclusions: Bcl-2 is upregulated in the pericontusional brain and CSF in the acute period after TBI. Bcl-2 has a neuroprotective role as a pro-survival protein in experimental models, and increased expression in patients can contribute to improvement in clinical outcomes. Its utility as a biomarker and therapeutic target to block neuronal apoptosis after TBI warrants further evaluation.

Chronic sub-anesthetic ketamine induces permanent hypolocomotion and impairment of hippocampus in adolescent cynomolgus monkeys

Ketamine has gained increasing popularity in adolescent drug abusers worldwide. However, relatively little is known about the long-term effects of recreational ketamine on adolescent hippocampus. The present study investigates the effects of different periods (1, 3 and 6 months) of recreational ketamine administration on locomotor activity and neuron damage in the hippocampus of adolescent cynomolgus monkeys. 32 4-year-old male cynomolgus monkeys were divided into control, 1-month, 3-month and 6-month groups. All animals in ketamine groups received daily intravenous injection with 1 mg/kg ketamine in saline for respective 1, 3 or 6 months while control group received normal saline. Automatic behaviors were recorded for 10 min before and after ketamine and saline administration. Meanwhile, the markers of apoptosis in the hippocampus were assessed using terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL), electron microscopy and western blotting. Results showed that ketamine significantly decreased locomotor activity, increased apoptotic neurons and pro-apoptotic proteins, cleaved Caspase-3 and Bax, while decreased the anti-apoptotic protein Bcl-2 in the hippocampus after 6-month ketamine administration. Our study suggested that chronically recreational ketamine might induce hypolocomotion and neurotoxic effect via apoptotic pathway in adolescent hippocampus of monkeys.

In utero transplantation of neural stem cells ameliorates maternal inflammation-induced prenatal white matter injury.

Prenatal white matter injury is a serious problem due to maternal inflammation leading to postnatal disabilities. In this study, we used the periventricular leukomalacia (PVL) model as a common prenatal white matter injury by maternal administration of lipopolysaccharide (LPS). Neural stem cells (NSCs) have shown therapeutic ability in neurological disorders through a different mechanism such as immunomodulation. Here, we studied the preventive potential of NSCs following in utero transplantation into the embryonic lateral ventricle in an LPS-induced white matter injury model. Pregnant animals were divided into three groups and received phosphate buffered saline, LPS, or LPS + NSCs. The brains of offspring were obtained and evaluated by real-time polymerase chain reaction (PCR), immunohistochemy, enzyme-linked immunosorbent assay (ELISA), terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL), and caspase-3 activity assay. The LPS-induced maternal inflammation degenerated the myelin sheath in the offspring periventricular region which was associated with an increased microglial number, oligodendrocytes degeneration, proinflammatory cytokine secretion, and cell apoptosis. The transplanted NSCs homed into the brain and ameliorated the evaluated parameters. The expression of proinflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), cell apoptosis and caspase-3 activity were inhibited by NSCs. In addition, Olig2 and myelin basic protein immunohistochemy staining showed that prenatal NSCs transplantation augmented the myelination in the periventricular white matter of offspring. In conclusion, we think that prenatal therapeutic strategies, such as in utero NSCs transplantation, may prevent prenatal white matter injury after birth.

Remote limb preconditioning protects against ischemia-induced neuronal death through ameliorating neuronal oxidative DNA damage and parthanatos

Remote limb preconditioning (RPC) ameliorates ischemia-induced cerebral infarction and promotes neurological function recovery; however, the mechanism of RPC hasn't been fully understood, which limits its clinical application. The present study aimed at exploring the underlying mechanism of RPC through testing its effects on neuronal oxidative DNA damage and parthanatos in a rat focal cerebral ischemia model. Infarct volume was investigated by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and neuronal survival was evaluated by Nissl staining. Oxidative DNA damage was investigated via analyzing the expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG). Besides, terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) and DNA laddering were utilized to evaluate neuronal DNA fragmentation. Moreover, we tested whether RPC regulated poly(ADP-ribose) polymer (PAR) and apoptosis inducing factor (AIF) pathway; thus, PAR expression, AIF translocation and AIF/histone H2AX (H2AX) interaction were investigated. The results showed that RPC exerted neuroprotective effects by ameliorating oxidative DNA damage and neuronal parthanatos; additionally, RPC suppressed PAR/AIF pathway through reducing AIF translocation and AIF/H2AX interaction. The present study further exposed neuroprotective mechanism of RPC, and provided new evidence for the research on RPC and ICS.

KHSRP Participates in Manganese-Induced Neurotoxicity in Rat Striatum and PC12 Cells

Manganese (Mn) is an essential micronutrient. However, exposure to high doses of Mn may lead to a neurological disease known as manganism, which is characterized by marked brain neuronal loss. K-homology splicing regulator protein (KHSRP) is a multifunctional RNA-binding protein and has been implicated in the regulation of multiple cellular signaling associated with neuronal apoptosis and survival, such as p38 mitogen-activated protein kinase (MAPK), nuclear factor kappaB (NF-κB), and Wnt/β-catenin pathways. In the present study, the role of KHSRP in Mn-induced neurotoxicity was investigated in vivo using a rat model of chronic Mn exposure and in vitro using differentiated PC12 cell cultures. Western blot and immunohistochemical analyses showed a significant upregulation of KHSRP in rat striatum following Mn exposure. Immunofluorescent labeling indicated that KHSRP was localized mainly in neurons. Terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick end labeling (TUNEL) assay showed that KHSRP was mainly distributed in apoptotic neurons. Increased KHSRP expression was positively correlated with the upregulation of several apoptosis-related proteins, such as p53, bax, and active caspase-3. In addition, significant co-localization of KHSRP and active caspase-3 in neurons after Mn exposure was also observed, suggesting a potential involvement of KHSRP in the regulation of Mn-induced striatal neuronal apoptosis. Importantly, interference with KHSRP apparently decreased the level of p53 and attenuated Mn-induced neuronal apoptosis. Taken together, these results indicate that upregulation of KHSRP may be involved in the pathological process underlying Mn neurotoxicity via the modulation of p53 signaling.

The expression changes of myelin and lymphocyte protein (MAL) following optic nerve crush in adult rats retinal ganglion cells.

Myelin and lymphocyte protein (MAL), a component of compact myelin, is highly expressed in oligodendrocytes and Schwann cells. It has been reported that MAL may play a vital role in the process of neuronal apoptosis following acute spinal cord injury. However, acquaintance regarding its distribution and possible function in the retina is limited. Therefore, in a rodent model of optic nerve crush (ONC), the dynamic changes of MAL in retina was detected. The expression of MAL was mainly located in the retinal ganglion cells (RGCs) and was increased strongly after ONC. The peak of MAL expression appeared on the third day. In addition, there was a concomitant upregulation of active-caspase-3, which also co-localized with MAL in RGCs. Moreover, co-localization of MAL with terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling (TUNEL) was detected in RGCs after ONC. Collectively, all these results suggested that the upregulation of MAL might play an important role in the pathophysiology of RGCs after ONC.

Chronic ketamine exposure induces permanent impairment of brain functions in adolescent cynomolgus monkeys

Ketamine, a non-competitive N-methyl-D-aspartic acid receptor antagonist, has emerged as an increasingly popular drug among young drug abusers worldwide. Available evidence suggests that ketamine produces acute impairments of working, episodic and semantic memory along with psychotogenic and dissociative effects when a single dose is given to healthy volunteers. However, understanding of the possible chronic effects of ketamine on behavior, cognitive anomalies and neurochemical homeostasis is still incomplete. Although previous human studies demonstrate that ketamine could impair a range of cognitive skills, investigation using non-human models would permit more precise exploration of the neurochemical mechanisms which may underlie the detrimental effects. The current study examined the abnormalities in behavior (move, walk, jump and climb) and apoptosis of the prefrontal cortex using terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) and apoptotic markers, including Bax, Bcl-2 and caspase-3 in adolescent male cynomolgus monkeys (Macaca fascicularis) after 1 or 6 months of sub-anesthetic ketamine administration (1 mg/kg, i.v.). Results showed that ketamine decreased locomotor activity and increased cell death in the prefrontal cortex of monkeys with 6 months of ketamine treatment when compared with the control monkeys. Such decreases were not found in the 1-month ketamine-treated group. Our study suggested that ketamine administration of recreational dose in monkeys might produce permanent and irreversible deficits in brain functions due to neurotoxic effects, involving the activation of apoptotic pathways in the prefrontal cortex.

Kainic Acid-Induced Neuronal Degeneration in Hippocampal Pyramidal Neurons Is Driven by Both Intrinsic and Extrinsic Factors: Analysis of FVB/N↔C57BL/6 Chimeras

The excitotoxic effects of kainic acid (KA) in the mouse hippocampus is strain dependent. Following KA administration, the large majority of hippocampal pyramidal cells die in the FVB/N (FVB) mouse, while the pyramidal cells of the C57BL/6 (B6) strain are largely spared. We generated aggregation chimeras between the sensitive FVB and the resistant B6 strains to investigate whether intrinsic or extrinsic features of a neuron confer cell vulnerability or resistance to KA. The constitutive expression of transgenic green fluorescence protein (GFP) or β-galactosidase expressed from the ROSA26 locus was used to mark cells in FVB or B6 mice, respectively. These makers enable the identification of cells from each parental genotype while TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling)-staining labeled dying cells. The analysis of the percentage of dying cells in FVB-GFP ↔ B6-ROSA chimeras yielded an intriguing mix of both intrinsic and extrinsic factors in the readout of cell phenotype. Thus, normally resistant B6-ROSA pyramidal neurons demonstrated an increasing sensitivity to KA, in a linear fashion, when the percentage of FVB-GFP cells was increased, either across chimeras or in different regions of the same chimera. However, the death of B6-ROSA pyramidal cells never exceeded ∼70% of the total amount of B6 neurons regardless of the amount of FVB cells in the chimeric hippocampus. In a similar manner, FVB-GFP cells show lower amounts of cell death in chimeras that are colonized by B6-ROSA cells, but again, are never fully rescued. These data indicate that both intrinsic and extrinsic factors modulate the sensitivity of hippocampal pyramidal cells to kainic acid.

Age‐related changes in cell dynamics of the postnatal mouse olfactory neuroepithelium: Cell proliferation, neuronal differentiation, and cell death

Age-related changes in cell proliferation, neuronal differentiation, and cell death in mouse olfactory neuroepithelium were investigated. Mice at the age of 10 days through 16 months were given a single injection of bromodeoxyuridine (BrdU). The olfactory mucosae were fixed at 9 timepoints ranging from 2 hours to 3 months after the injection and examined using double immunostaining for BrdU and olfactory marker protein (OMP), and double staining with terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) and immunostaining for OMP. The number of BrdU-labeled cells/mm epithelial length initially increased, peaked at 2-3 days after the BrdU injection, then declined at each age. The number of BrdU- and TUNEL-labeled neuronal cells both decreased with increasing age, suggesting that the rates of both cell proliferation and cell death in the olfactory neuroepithelium decrease with increasing age. Double-labeled cells for BrdU and OMP appeared at 7 days after injection in all age groups, suggesting that the time required for neuronal differentiation is broadly similar irrespective of age. In older age groups, smaller amounts of the newly produced cohort are integrated into the OMP-positive ORN population, and even once it is integrated it is eliminated from the population more rapidly compared to the younger age groups. Furthermore, TUNEL assay showed that the fraction of apoptotic cells distributed in the OMP-positive layer/total apoptotic cells decreased with age. This observation suggests that the turnover of mature ORNs is slower in the older neuroepithelium compared to the younger neuroepithelium.

NMDA Receptor Function and NMDA Receptor-Dependent Phosphorylation of Huntingtin Is Altered by the Endocytic Protein HIP1

Huntingtin-interacting protein 1 (HIP1) is an endocytic adaptor protein that plays a role in clathrin-mediated endocytosis and the ligand-induced internalization of AMPA receptors (AMPARs) (Metzler et al., 2003). In the present study, we investigated the role of HIP1 in NMDA receptor (NMDAR) function by analyzing NMDA-dependent transport and NMDA-induced excitotoxicity in neurons from HIP1-/- mice. HIP1 colocalizes with NMDARs in hippocampal and cortical neurons and affinity purifies with NMDARs by GST (glutathione S-transferase) pull down and coimmunoprecipitation. A profound decrease in NMDA-induced AMPAR internalization of 75% occurs in neurons from HIP1-/- mice compared with wild type, using a quantitative single-cell-based internalization assay. This defect in NMDA-dependent removal of surface AMPARs is in agreement with the observed defect in long-term depression induction in hippocampal brain slices of HIP1-/- mice and supports a role of HIP1 in AMPAR internalization in vivo. HIP1-/- neurons are partially protected from NMDA-induced excitotoxicity as assessed by LDH (lactate dehydrogenase) release, TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling) and caspase-3 activation assays, which points to a role of HIP1 in NMDA-induced cell death. Interestingly, phosphorylation of Akt and its substrate huntingtin (htt) decreases during NMDA-induced excitotoxicity by 48 and 31%, respectively. This decrease is significantly modulated by HIP1, resulting in 94 and 48% changes in P-Akt and P-htt levels in HIP1-/- neurons, respectively. In summary, we have shown that HIP1 influences important NMDAR functions and that both HIP1 and htt participate in NMDA-induced cell death. These findings may provide novel insights into the cellular mechanisms underlying enhanced NMDA-induced excitotoxicity in Huntington's disease.

Neurogenesis, migration, and apoptosis in the vomeronasal epithelium of adult mice

The location of neurogenesis and the direction of migration of neurons in the adult mouse vomeronasal organ is controversial. Cell division occurs at the center, and particularly, at the edges of the epithelium. Newly generated cells at the center of the epithelium participate in neurogenesis, however, it is unknown to what extent dividing cells at the edges participate in growth, become apoptotic or mature into neurons. Premitotic cells were labeled with bromodeoxyuridine (BrdU) in adult mice and animals allowed to survive for different postinjection periods. The terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling (TUNEL) method was used to show the distribution of apoptotic cells. The vertical and horizontal position of BrdU-labeled cells was analyzed as a function of postinjection survival time. Vertical and horizontal migration of BrdU-labeled cells were detected. Cells in the central portions of the epithelium migrated vertically to become neurons as demonstrated by co-expression of olfactory marker protein. Cells at the edges migrated horizontally very slowly (less than 10% of the distance from the edge to the center of the epithelium per month), thus indicating that these cells participate in cell renewal exclusively in marginal regions. Neural turnover in the mouse vomeronasal epithelium, therefore appears to occur through a process of vertical migration. Data on the distribution of apoptotic cells indicate that a number of dividing cells throughout the epithelium, but particularly at the edges, die before becoming functional neurons. Accordingly, most dividing cells at the edges probably constitute a reservoir of stem cells dying before differentiation.

Moderate Zinc Deficiency Increases Cell Death After Brain Injury in the Rat

Zinc supplementation has been used clinically to reduce Zn losses and protein turnover in patients suffering from traumatic brain injury. Despite the known role of zinc in cell survival and integrity, the influence of zinc status on central nervous system wound healing in the weeks and months after brain injury has not been addressed. In this investigation, we examined cell death after unilateral cortical stab wounds in adult rats (n=5 per group) that were provided diets containing adequate zinc (30 mg Zn/kg diet), supplemental zinc (180 mg/kg), or moderately deficient zinc (5 mg/kg). Four weeks following the brain injury there was a 1.82-2.65-fold increase in terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick-end labeling (TUNEL)-positive cells with DNA fragmentation at the site of injury in animals receiving a moderately zinc deficient diet compared to animals receiving a zinc-adequate or supplemented diet (p#0.05). Examination of the nuclear morphology of these cells suggested the presence of both apoptosis and necrosis. Immunohistochemistry showed that the TUNEL-positive cells expressed both ED-1 and OX-42, identifying them as microglia/macrophages. Thus it appears that adequate zinc status may be necessary to minimize the amount of neuroimmune cell death after brain injury.

Traumatic Brain Injury Alters the Molecular Fingerprint of TUNEL-Positive Cortical NeuronsIn Vivo: A Single-Cell Analysis

The cerebral cortex is selectively vulnerable to cell death after traumatic brain injury (TBI). We hypothesized that the ratio of mRNAs encoding proteins important for cell survival and/or cell death is altered in individual damaged neurons after injury that may contribute to the cell's fate. To investigate this possibility, we used amplified antisense mRNA (aRNA) amplification to examine the relative abundance of 31 selected candidate mRNAs in individual cortical neurons with fragmented DNA at 12 or 24 hr after lateral fluid percussion brain injury in anesthetized rats. Only pyramidal neurons characterized by nuclear terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) reactivity with little cytoplasmic staining were analyzed. For controls, non-TUNEL-positive neurons from the cortex of sham-injured animals were obtained and subjected to aRNA amplification. At 12 hr after injury, injured neurons exhibited a decrease in the relative abundance of specific mRNAs including those encoding for endogenous neuroprotective proteins. By 24 hr after injury, many of the mRNAs altered at 12 hr after injury had returned to baseline (sham-injured) levels except for increases in caspase-2 and bax mRNAs. These data suggest that TBI induces a temporal and selective alteration in the gene expression profiles or "molecular fingerprints" of TUNEL-positive neurons in the cerebral cortex. These patterns of gene expression may provide information about the molecular basis of cell death in this region after TBI and may suggest multiple avenues for therapeutic intervention.

Optimal Prefixation of Cells To Demonstrate Apoptosis by the TUNEL Method

To determine the optimal fixation method for cultured human ovarian cancer cell line SHIN-3 to document cisplatin-induced apoptosis by the terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end-labeling (TUNEL) assay. Cisplatin-treated cancer cell suspensions were (1) fixed in 4% buffered formalin for 10 minutes (BF group); (2) treated with 2% Carbowax in 50% ethanol (CW) for 10 minutes and then fixed in 100% ethanol for one hour (CW + ET); or (3) treated with CW for 10 minutes and then fixed in 4% buffered formalin for one hour (CW + BF). Cell morphology, adhesion to the glass slides and TUNEL reactivity were compared among the three groups. The effects of prolonged prefixation of cell suspensions in CW and of the postfixation of cell smears in BF for one, three and seven days were also examined. CW + BF treatment yielded satisfactory cell morphology, minimum cell loss and an excellent TUNEL reaction. However, prolonged prefixation in CW resulted in cell shrinkage. CW + BF treatment can be widely recommended for use with cytologic preparations for the TUNEL assay.