Biomedical subjects

W Pei

Publications and source records attributed to W Pei.

At least 19 recordsLinked to original sources

PubMed2006

Two new reactive targets of 2,5-hexanedione in vitro - beta-alanine and glycine.

In this study, we found that two amino acids reacted with 2,5-hexanedione to form new reaction products in vitro, respectively. In the reaction of beta-alanine and 2,5-hexanedione, a reaction product was obtained and analyses of obtained results showed it was 3-(2,5-dimethyl-1H-pyrrol-1-yl)propanoic acid; in the reaction of glycine and 2,5-hexanedione, a reaction product was also obtained and analyses showed it was (2,5-dimethyl-1H-pyrrol-1-yl)acetic acid. Two reaction products were found to be oxidized easily; in addition, the latter was more easily to be oxidized than the former in the air. Our discoveries demonstrated that reactions between amino acids and 2,5-hexanedione could exist possibly in vitro. At present, it is clear that 2,5-hexanedione causes either axon atrophy or swelling, but the underlying molecular mechanism is still unclear. Since both beta-alanine and glycine are considered as neurotransmitter in the central nervous system, the reaction products remain to be identified in vivo.

Amino Acids↗

PubMed2003

Proliferation, differentiation and self-renewal of osteoprogenitors in vertebral cell populations from aged and young female rats.

A significant contribution to the bone loss associated with aging is likely to be a decline in bone formation. We have characterized and compared the number, capacity for proliferation and differentiation and the self-renewal ability of osteoprogenitors of aged (17-26-month-old) and young (1.5-month-old) female Wistar rats using limiting dilution analyses and continuous subculture experiments. Cells were obtained from outgrowths of explants of lumbar vertebrae (L1-L6) and grown in alpha-minimal essential medium (alpha-MEM), 10% FBS and 50 microg/ml ascorbic acid with or without dexamethasone (Dex; 0.3-300 nM) or progesterone (Prog; 0.01-10 microM). Growth curves for cell populations of both age groups were similar with population doubling times of 27.1 and 26.7 h for the aged and young animals, respectively. Osteoprogenitors from both age groups formed bone nodules when cultured in the presence of either Dex or Prog. Limiting dilution analysis in the presence of 10 nM Dex showed no difference between the aged and young rats in the number of colony forming units-fibroblast (CFU-F), alkaline phosphatase-positive colony forming units-fibroblast (AP+ CFU-F) or colony forming units-osteoblast (CFU-O). No differences were also found for any progenitor within the aged group. Limiting dilution analysis in the presence of 3 microM Prog showed no differences in the numbers of CFU-F, AP+ CFU-F or CFU-O between the aged and young groups or within the aged group. Continuous subculture of cells in the presence of 10 nM Dex revealed that the number of nodules per 10(4) plated cells increased in second subculture over first subculture cells in the young group but decreased in the aged group. Also, in third to fifth subculture cells, the number of nodules was lower in the aged group than in the young group. A similar pattern was observed in the presence of 3 microM Prog. Results indicate that the cell population doubling times, growth characteristics, and the number of CFU-F and osteoprogenitors in vertebral bone cell populations from aged rats and young rats are similar. This suggests that the bone loss associated with aging is not caused by a decrease in osteoprogenitor cell number. However, cell populations from the aged rats showed a reduced capacity for self-renewal in vitro, which would ultimately translate into a reduced number of osteoblasts and might be partly responsible for a decrease in bone formation in aged animals.

Aging↗

PubMed2002

Identification of dexamethasone-dependent osteoprogenitors in cell populations derived from adult human female bone.

The purpose of this investigation was to establish whether or not dexamethasone (Dex)-dependent osteoprogenitors with sufficient proliferative capacity to form a colony of bone-forming osteoblasts could be identified in cell populations isolated from adult human bone. This question is relevant because of the ongoing controversy regarding the effects of dexamethasone on bone formation in humans, the clearly different effects of dexamethasone on osteoprogenitor differentiation in mouse vs. rat bone cell populations, and the related question of whether observations in either rat or mouse systems are applicable to human systems. To answer the question, we isolated cell populations from distal femoral cancellous bone of 8 female patients with osteoarthritis and quantitated the number of Dex-dependent osteoprogenitors in these populations by counting the number of osteoblastic colonies forming bone (bone nodules) or unmineralized bone matrix (osteoid nodules). Dex increased alkaline phosphatase (AP) content in all populations, induced bone nodule formation in 2 of the 8 populations, and induced formation of AP-positive clusters of cells with osteoblastic morphology in one. Treatment with 1,25-dihydroxyvitamin D3 increased osteocalcin (OC) production in the nodule forming populations, but not in the non-nodule-forming populations. Our results thus establish that Dex-dependent osteoprogenitors with sufficient proliferative capacity to form bone or osteoid nodules are present in cell populations derived from adult human bone. They also show that frozen primary human bone cell populations that have been characterized previously in terms of the number of Dex-dependent osteoprogenitors present can be used to further study the characteristics of such progenitors.

Adult↗

PubMed2001

Ion dynamics in steady collisionless driven reconnection.

Steady collisionless driven reconnection in an open system is investigated by means of a new two-dimensional full-particle simulation. The reconnection rate is controlled by an external driving electric field. Ion-meandering motion plays an important role in ion dynamics which controls the spatial structures of ion quantities. Although the electric current is predominantly carried by electrons, the current layer has the half-width of the ion-meandering orbit scale because the density profile is controlled by massive-ion motion. Thus, the global dynamic behavior of reconnection is dominantly controlled by ion dynamics. An electrostatic field generated through the finite-Larmor-radius effect leads to electron acceleration in the equilibrium current direction in the ion-dissipation region and ion heating by intensifying meandering motion. Our results are in agreement with the recent experimental results of Yamada et al. [Phys. Plasmas 7, 1781 (2000)] and of Hus et al. [Phys. Rev. Lett. 84, 3859 (2000)].

Journal Article↗

PubMed2001

Downregulation of matrix metalloproteinases and reduction in collagen damage in the failing human heart after support with left ventricular assist devices.

BACKGROUND: Left ventricular assist device (LVAD) support of the failing heart induces salutary changes in myocardial structure and function. Matrix metalloproteinases (MMPs) are increased in the failing heart and are induced by stretch in cardiac cells in vitro. We hypothesized that mechanical unloading may affect LV plasticity by regulating MMPs and their substrates. METHODS AND RESULTS: LV samples were collected from patients with dilated cardiomyopathy (DCM, n=14) or ischemic cardiomyopathy (ICM, n=16) at the time of implantation of the LVAD and again during cardiac transplantation. MMP-1, -3, and -9 were measured by ELISA, MMP-2 and -9 gelatinolytic activity by gelatin zymography, and tissue inhibitors of metalloproteinases (TIMPs) by Western blot. Total soluble and insoluble collagens were separated by pepsin solubilization, and the contents were determined by quantification of hydroxyproline. The undenatured soluble collagen was measured by Sircol collagen assay. The results showed that MMP-1 and -9 were decreased, whereas TIMP-1 and -3 were increased, but there was no change in MMP-2 and -3 and TIMP-2 and -4 after LVAD support. The undenatured collagen was increased, with the ratio of undenatured to total soluble collagens increased in ICM and that of insoluble to total soluble collagens increased in DCM after LVAD support. CONCLUSIONS: The reduced MMPs and increased TIMPs and ratios of undenatured to total soluble collagens and insoluble to total soluble collagens after LVAD support suggest that reduced MMP activity diminished damage to the matrix. These changes may contribute to the functional recovery and LV plasticity after LVAD support.

Adult↗

PubMed2001

Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures.

Nuclear changes, including internucleosomal DNA fragmentation, are characteristic features of neuronal apoptosis resulting from transient cerebral ischemia and related brain insults for which the molecular mechanism has not been elucidated. Recent studies suggest that a caspase-3-mediated mechanism may be involved in the process of nuclear degradation in ischemic neurons. In this study, we cloned from rat brain a homolog cDNA encoding caspase-activated deoxyribonuclease (CAD)/DNA fragmentation factor 40 (DFF40), a 40 kDa nuclear enzyme that is activated by caspase-3 and promotes apoptotic DNA degradation. Subsequently, we investigated the role of CAD/DFF40 in the induction of internucleosomal DNA fragmentation in the hippocampus in a rat model of transient global ischemia and in primary neuronal cultures under ischemia-like conditions. At 8-72 hr after ischemia, CAD/DFF40 mRNA and protein were induced in the degenerating hippocampal CA1 neurons. CAD/DFF40 formed a heterodimeric complex in the nucleus with its natural inhibitor CAD (ICAD) and was activated after ischemia in a delayed manner (>24 hr) by caspase-3, which translocated into the nucleus and cleaved ICAD. Furthermore, an induced CAD/DFF40 activity was detected in nuclear extracts in both in vivo and in vitro models, and the DNA degradation activity of CAD/DFF40 was inhibited by purified ICAD protein. These results strongly suggest that CAD/DFF40 is the endogenous endonuclease that mediates caspase-3-dependent internucleosomal DNA degradation and related nuclear alterations in ischemic neurons.

Animals↗

PubMed2001

Intracellular Bax translocation after transient cerebral ischemia: implications for a role of the mitochondrial apoptotic signaling pathway in ischemic neuronal death.

Activation of terminal caspases such as caspase-3 plays an important role in the execution of neuronal cell death after transient cerebral ischemia. Although the precise mechanism by which terminal caspases are activated in ischemic neurons remains elusive, recent studies have postulated that the mitochondrial cell death-signaling pathway may participate in this process. The bcl-2 family member protein Bax is a potent proapoptotic molecule that, on translocation from cytosol to mitochondria, triggers the activation of terminal caspases by increasing mitochondrial membrane permeability and resulting in the release of apoptosis-promoting factors, including cytochrome c. In the present study, the role of intracellular Bax translocation in ischemic brain injury was investigated in a rat model of transient focal ischemia (30 minutes) and reperfusion (1 to 72 hours). Immunochemical studies revealed that transient ischemia induced a rapid translocation of Bax from cytosol to mitochondria in caudate neurons, with a temporal profile and regional distribution coinciding with the mitochondrial release of cytochrome c and caspase-9. Further, in postischemic caudate putamen in vivo and in isolated brain mitochondria in vitro, the authors found enhanced heterodimerization between Bax and the mitochondrial membrane permeabilization-related proteins adenine nucleotide translocator (ANT) and voltage-dependent anion channel. The ANT inhibitor bongkrekic acid prevented Bax and ANT interactions and inhibited Bax-triggered caspase-9 release from isolated brain mitochondria in vitro. Bongkrekic acid also offered significant neuroprotection against ischemia-induced caspase-3 and caspase-9 activation and cell death in the brain. These results strongly suggest that the Bax-mediated mitochondrial apoptotic signaling pathway may play an important role in ischemic neuronal injury.

Animals↗

PubMed2000

Characterization of the rat DNA fragmentation factor 35/Inhibitor of caspase-activated DNase (Short form). The endogenous inhibitor of caspase-dependent DNA fragmentation in neuronal apoptosis.

Nuclear changes, including internucleosomal DNA fragmentation, are classical manifestations of apoptosis for which the biochemical mechanisms have not been fully elucidated, particularly in neuronal cells. We have cloned the rat DNA fragmentation factor 35/inhibitor of caspase-activated DNase (short form) (DFF35/ICAD(S)) and found it to be the predominant form of ICAD present in rodent brain cells as well as in many other types of cells. DFF35/ICAD(S) forms a functional complex with DFF40/caspase-activated DNase (CAD) in the nucleus, and when its caspase-resistant mutant is over-expressed, it inhibits the nuclease activity, internucleosomal DNA fragmentation, and nuclear fragmentation but not the shrinkage and condensation of the nucleus, in neuron-differentiated PC12 cells in response to apoptosis inducers. DFF40/CAD is found to be localized mainly in the nucleus, and during neuronal apoptosis, there is no evidence of further nuclear translocation of this molecule. It is further suggested that inactivation of DFF40/CAD-bound DFF35 and subsequent activation of DFF40/CAD during apoptosis of neuronal cells may not occur in the cytosol but rather in the nucleus through a novel mechanism that requires nuclear translocation of caspases. These results establish that DFF35/ICAD(S) is the endogenous inhibitor of DFF40/CAD and caspase-dependent apoptotic DNA fragmentation in neurons.

Amino Acid Sequence↗

PubMed2000

Detection of DNA base-excision repair activity for oxidative lesions in adult rat brain mitochondria.

Endogenous oxidative damage to brain mitochondrial DNA and consequential disturbances of gene expression and mitochondrial dysfunction have long been implicated in aging and the pathogenesis of neurodegenerative diseases. It has yet to be determined, however, whether mitochondria in brain cells contain an active DNA repair system and, if so, how this system functions. Therefore, the capacity for the repair of defined types of oxidative DNA lesions has been investigated in adult rat brain mitochondria. Using in vitro DNA incorporation repair assay, we have detected base excision repair (BER) activity for the common oxidative DNA adduct 8-hydroxyl-2'-deoxyguanine (8-oxodG) in mitochondria protein extracts from cortical tissues and cultured primary cortical neurons and astrocytes. The levels of BER activity were both protein concentration-dependent and repair-incubation time-dependent. To resolve the BER pathway, the activity of essential BER enzymes was examined in mitochondria using oligonucleotide incision assay, DNA polymerase assay, and DNA ligase assay employing specific DNA substrates. Mitochondrial extracts were able to remove specifically 8-oxodG, uracil, and the apurinic/apyrimidinic abasic site from substrates. Moreover, a gamma-like DNA polymerase activity and a DNA ligase activity were detected in mitochondiral extracts, based on the formation of specific repair products. These results demonstrate that adult brain mitochondria possess an active BER system for repairing oxidative DNA lesions. This repair system appears to function by sequential actions of DNA repair enzymes that are homologous to, but not identical to, that in the nucleus. Thus, BER may represent an endogenous protective mechanism against oxidative damage to mitochondrial, as well as nuclear, genomes in brain cells.

Animals↗

PubMed2000

Mass spectrometric study of gamma-aryl-alpha,-gamma-diethoxy-alpha, beta-butenolides.

Electron impact mass spectra of eight of the title compounds are reported. Abundant fragment ions were produced under electron impact (EI) conditions and, with one exception, the ($¿hbox¿ArC¿¿equiv$ O) ions were the base peaks. The EI fragmentation mechanisms of two representative compounds were studied with the aid of high-resolution and mass-analyzed ion kinetic energy spectrometry (MIKES) data. The M(+) ions fragment to give both an odd-electron ion and an even-electron fragment ion. Two H-atom rearrangements proceeding via four-membered ring intermediates and three losses of CO through i- and alpha-fragmentations were observed under EI. On comparing fragmentations under EI conditions with those under FAB conditions for two of the compounds, the fragmentation mechanisms were reasonably similar, with additional fragmentations rationalized in terms of the ionization proton being located on the oxygen atom of the beta-ethoxy group.

4-Butyrolactone↗

PubMed2000

Support for linkage of familial combined hyperlipidemia to chromosome 1q21-q23 in Chinese and German families.

We examined familial combined hyperlipidemia (FCHL) families from nonisolated regions in Germany and China to see if we could corroborate support for a chromosome 1q FCHL locus in more general populations. We recruited 24 German families with 137 members, 92 of whom met the criteria of affected in terms of the low density lipoprotein (LDL) and triglyceride levels in excess of the 90th percentile for age and gender. In China, we recruited 12 families with a total of 81 members. All affected persons had total cholesterol concentrations >240 mg/dl and triglyceride concentrations >250 mg/dl. We examined the markers APOA2, D1S1677, D1S104, D1S194, D1S426, and D1S196. Two-point linkage analysis allowing for heterogeneity gave a maximum linkage of disorder score (HLOD) of 2.60 right over D1S194, estimating the proportion of linked families at 36%. This marker is adjacent to D1S104. The evidence for linkage was roughly the same both in the German (HLOD 1.40) and Chinese families (HLOD 1.52). Marker D1S194 is close to the retinoid X receptor (RXR) gene locus, which was found to be linked to triglyceride levels in an earlier twin study from our laboratory. We interpret our observations as encouraging support for the recent findings indicating the presence of a gene for FCHL on chromosome 1q. Furthermore, since DIS194 is adjacent to the gene for the RXR, we suggest that RXR is an attractive candidate for involvement in FCHL.

Adult↗

PubMed2000

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia.

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.

Animals↗

PubMed2000

Overexpression of the cell death suppressor Bcl-w in ischemic brain: implications for a neuroprotective role via the mitochondrial pathway.

Bcl-w is a newly described cell death suppressor member of the Bcl-2 gene family. As these genes may have a role in the outcome of ischemic brain injury, the regional expression of Bcl-w protein in rat brain was examined at 6 to 72 hours after 90 minutes of transient middle cerebral artery occlusion. Bcl-w protein, although constitutively expressed at low levels in nonischemic brain, was found to be overexpressed in ischemic brain at all time points studied. Up-regulation of Bcl-w protein was particularly abundant in the penumbral region of the cortex and mainly in cells lacking DNA fragmentation. In the cortical penumbra, Bcl-w protein was detected predominantly in neurons and showed mitochondrial localization, as determined using double-label immunohistochemistry. Bcl-w expression was also detectable, to a lesser extent, in reactive astrocytes in the infarct border zone and in microvessel walls in the infarct regions. At the mechanistic level, incubation of isolated brain mitochondria with the addition of recombinant Bax or high concentration of calcium resulted in release of cytochrome c from the mitochondria. In the presence of recombinant Bcl-w protein, however, the release of cytochrome c induced by Bax or calcium was largely inhibited. Further, recombinant Bcl-w protein inhibited calcium-induced loss of mitochondrial transmembrane potential, indicative of permeability transition, in a dose-dependent manner. These results suggest that Bcl-w may be an endogenous neuroprotectant against ischemic neuronal death and that, like its analogues such as Bcl-2 and Bcl-x-long, Bcl-w may achieve this protection via the mitochondrial death-regulatory pathway.

Animals↗

PubMed2000

Angiotensin-converting enzyme gene insertion/deletion (I/D) polymorphism in hypertensive patients with different degrees of obstructive sleep apnea.

To investigate the role of the angiotensin-converting enzyme gene (ACE) insertion (I)/deletion (D) polymorphism in hypertensive patients with different degrees of obstructive sleep apnea (OSA). A case-control study was performed. One hundred seventy four Chinese subjects were divided into four groups depending on the severity of OSA as follows: 1) normal control group (NC, n=68), 2) isolated hypertension group (HT, n=45), 3) hypertensive patients with mild OSA group (MO, n=27), and 4) hypertensive patients with moderate to severe OSA group (MSO, n=34). The distribution of ACE gene I/D allele and genotypes were analyzed in the subject population, as was an OSA pedigree. The study showed that the frequency of ACE gene I/D polymorphism differed significantly among the four groups. The frequency of I allele and II genotype were significantly higher in the MSO group than in the other groups (p<0.05). The distribution of I allele and II genotype showed no significant difference between any of the other groups (p>0.05, respectively). Meanwhile the higher frequency of I allele and II genotype was observed in the OSA pedigree. The higher frequency of ACE gene I allele and II genotype were closely associated with the hypertensive patients with MSO. The inherited factors played an important role in the pathogenesis of hypertensive patients with MSO.

Adult↗

PubMed2000

[Linkage of familial combined hyperlipidemia to chromosome 1q21-23 in Chinese and German families].

OBJECTIVE: To examine familial combined hyperlipidemia (FCHL) families from nonisolated regions in China and Germany to see if we could corroborate support for a chromosome 1q FCHL locus in more general populations. METHODS: We recruited 24 German families with 133 members and 12 Chinese families with a total of 81 members in China. The markers ApoA2, D1S1677, D1S104, and D1S194 were examined by multipoint linkage analysis. RESULTS: Multipoint linkage analysis allowing for heterogeneity gave a maximum LOD score (HLOD) of 1.97 right over D1S194, estimating the proportion of linked families at 17%. This marker was adjacent to D1S104. The evidence for linkage was roughly the same both in the German (13%, HLOD(D1S194) = 1.08) and Chinese families (proportion of linked families 26%, HLOD(D1S194) = 0.97). CONCLUSION: In the light of the relatively small numbers and the heterogeneity our populations represent, we interpret our observations as encouraging support for the recent findings indicating the presence of linkage for FCHL on chromosome 1q21-23.

Apolipoprotein A-II↗

PubMed1997

Ubiquitin C-terminal hydrolase is an immediate-early gene essential for long-term facilitation in Aplysia.

The switch from short-term to long-term facilitation of the synapses between sensory and motor neurons mediating gill and tail withdrawal reflexes in Aplysia requires CREB-mediated transcription and new protein synthesis. We isolated several downstream genes, one of which encodes a neuron-specific ubiquitin C-terminal hydrolase. This rapidly induced gene encodes an enzyme that associates with the proteasome and increases its proteolytic activity. This regulated proteolysis is essential for long-term facilitation. Inhibiting the expression or function of the hydrolase blocks induction of long-term but not short-term facilitation. We suggest that the enhanced proteasome activity increases degradation of substrates that normally inhibit long-term facilitation. Thus, through induction of the hydrolase and the resulting up-regulation of the ubiquitin pathway, learning recruits a regulated form of proteolysis that removes inhibitory constraints on long-term memory storage.

Amino Acid Sequence↗

PubMed1997

Electrophoretic demonstration of high molecular weight fibrin degradation products persisting in chronic subdural hematomas.

Local hyperfibrinolysis plays an important role in the pathogenesis of chronic subdural hematoma (CSH). The purpose of this study is to elucidate the nature of the local hyperfibrinolysis in CSH, by comparing the pattern of fibrin/fibrinogen degradation product (FDP) of hematomas with that of purified fibrin clots digested by plasmin in vitro. Forty-seven hematoma samples were subjected to the analysis. FDPs of CSH and the purified fibrin clots digested by plasmin for different incubation times were identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting, and then quantified by densitometry. The effect of alpha-2 plasmin inhibitor (A2-PI) on the degradation process was also studied. The FDP pattern of CSH was similar to those of purified fibrin digested by plasmin for 0.75-2 h. The FDP composition of CSH was closest to that of purified fibrin digested for 1 h. By adding the physiological concentration of A2-PI at the second hour, further degradation of high molecular weight FDPs was inhibited and the early FDP pattern persisted after 24 h. Local hyperfibrinolysis in CSHs is characterized by incomplete fibrinolysis, which occurs only on the solid fibrin clot, and is arrested in the liquid hematoma. As a result, high molecular weight FDPs persist in CSHs for weeks or months in the hematoma. A2-PI seems to play an important role in producing this unique FDP pattern in CSH.

Chronic Disease↗

PubMed1997

Early detection of DNA strand breaks in the brain after transient focal ischemia: implications for the role of DNA damage in apoptosis and neuronal cell death.

Using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL), we investigated the evolution of DNA strand breaks, a marker of DNA damage, in rat brain after 1 h of middle cerebral artery occlusion and various durations of reperfusion. DNA single-strand breaks (SSBs) detected by PANT were present in neurons after as little as 1 min of reperfusion. Numbers of neurons containing an SSB increased progressively in the ischemic core but decreased in the ischemic penumbra after 1 h of reperfusion. DNA double-strand breaks (DSBs) detected by TUNEL were first seen in neurons after 1 h of reperfusion, and their numbers then increased progressively in the ischemic core, with a regional distribution similar to that of SSBs. However, the number of SSB-containing cells was greater than that of DSB-containing cells at all time points tested. SSB-containing cells detected within the first hour of reperfusion were exclusively neuronal and exhibited normal nuclear morphology. At 16-72 h of reperfusion, many SSB- and DSB-containing cells, including both neurons and astrocytes, showed morphological changes consistent with apoptosis. Gel electrophoresis of DNA isolated from the ischemic core showed DNA fragmentation at 24 h, when both SSBs and DSBs were present, but not at 1 h, when few DSBs were detected. These results suggest that damage to nuclear DNA is an early event after neuronal ischemia and that the accumulation of unrepaired DNA SSBs may contribute to delayed ischemic neuronal death, perhaps by triggering apoptosis.

Animals↗