Edward Preston

Global ischemia can cause DNA fragmentation indicative of apoptosis in rat brain

Laddered DNA indicative of apoptosis was observed in the CA1 layer of hippocampus and in dorsolateral striatum following a global cerebral ischemic insult produced by transient two vessel occlusion in rats. The extent of this DNA damage was proportional to the duration of the ischemic episode. Breaks in DNA were demonstrated in situ in sections from post-ischemic brain in neurons of the hippocampal CA1 which undergo selective neuronal death but not in other cell types. It is concluded that there is an apoptotic component to selective neuronal death following global ischemia in rat brain.

Biphasic Opening of the Blood-Brain Barrier Following Transient Focal Ischemia: Effects of Hypothermia

OBJECTIVE Tracer constants (Ki) for blood-to-brain diffusion of sucrose were measured in the rat to profile the time course of blood-brain barrier injury after temporary focal ischemia, and to determine the influence of post-ischemic hypothermia. METHODS Spontaneously hypertensive rats were subjected to transient (2 hours) clip occlusion of the right middle cerebral artery. Reperfusion times ranged from 1.5 min to 46 hours, and i.v. 3H-sucrose was circulated for 30 min prior to each time point (1 h, 4 h, 22 h, and 46 h; n = 5-7 per time point). Ki was calculated from the ratio of parenchymal tracer uptake and the time-integrated plasma concentration. Additional groups of rats (n = 7-8) were maintained either normothermic (37.5 degrees C) or hypothermic (32.5 degrees C or 28.5 degrees C) for the first 6 hours of reperfusion, and Ki was measured at 46 hours. RESULTS Rats injected after 1.5-2 min exhibited a 10-fold increase in Ki for cortical regions supplied by the right middle cerebral artery (p < 0.01). This barrier opening had closed within 1 to 4 hours post-reperfusion. By 22 hours, the blood-brain barrier had re-opened, with further opening 22 and 46 hours (p < 0.01), resulting in edema. Whole body hypothermia (28 degrees C-29 degrees C) during the first six hours of reperfusion prevented opening, reducing Ki by over 50% (p < 0.05). CONCLUSIONS Transient middle cerebral artery occlusion evokes a marked biphasic opening of the cortical blood-brain barrier, the second phase of which causes vasogenic edema. Hypothermic treatment reduced infarct volume and the late opening of the blood-brain barrier. This opening of the blood-brain barrier may enhance delivery of low permeability neuroprotective agents.

Differences in DNA fragmentation following transient cerebral or decapitation ischemia in rats.

The time course of appearance of cells with DNA damage was studied in rats following transient severe forebrain ischemia. This DNA damage could be detected by in situ end-labeling on brain sections. The breaks in DNA appeared selectively by day 1 in the striatum and later in the CA1 region of the hippocampus. It was possible by double labeling to show that there was no DNA damage in astrocytes. The DNA breaks consisted of laddered DNA fragments indicative of an ordered apoptotic type of internucleosomal cleavage, which persisted without smearing for up to 7 days of reperfusion. In contrast, the DNA breaks following ischemia induced by decapitation were random and, after gel electrophoresis, consisted of smeared fragments of multiple sizes. There was some early regional cellular death, restricted to the dentate of the hippocampus, prior to the pannecrotic degeneration. It is concluded that transient forebrain ischemia leads to a type of neuronal destruction that is not random necrosis but that shares some component of the apoptotic cell death pathway.

Three openings of the blood-brain barrier produced by forebrain ischemia in the rat.

A sensitive radiotracer method was used to explore the time course and regional pattern of blood-brain barrier (BBB) opening that occurs in a rat forebrain ischemia model that mimics temporary cardiac arrest. Immediately following 10 min of ischemia, transfer constants (Ki) for blood to brain permeation of [3H]sucrose were augmented severalfold, indicating widespread BBB opening. After 6 h, a delayed intensification of opening was evident in striatum and hippocampus, regions known to undergo selective, delayed neuronal death. There was generalized BBB recovery by 24 h except in experiments that involved prolonged ischemia (25 min) or concomitant brain hyperthermia (41 degrees C, 10 min). These protocols evoked a third opening; a marked upward increment in Ki and % H2O developed in neocortex between 6 and 24 h post-ischemia. Pharmacological or other manipulations of these temporal and regional patterns of altered transfer constants may aid understanding of the interplay between microvascular damage, edema, and neuronal death following brain ischemia.

Translation-State Analysis of Gene Expression in Mouse Brain after Focal Ischemia

Confounding any genome-scale analysis of gene expression after cerebral ischemia is massive suppression of protein synthesis. This inefficient translation questions the utility of examining profiles of total transcripts. Our approach to such postischemic gene profiling in the mouse by microarray analysis was to concentrate on those mRNAs bound to polyribosomes. In our proof-of-principle study, polysomally bound and unbound mRNAs were subjected to microarray analysis: of the 1,161 transcripts that we found to increase after ischemia, only 36% were bound to polyribosomes. In addition to the expected increases in heat-shock proteins and metallothioneins, increases in several other bound transcripts involved in the promotion of cell survival or antiinflammatory behavior were noted, such as CD63 (Lamp3), Lcn2 (lipocalin-2), Msn (moesin), and UCP2 (uncoupling protein 2), all of which showed increases in cognate protein by Western blotting. The list of heretofore nonfunctionally annotated transcripts (RIKEN clones/ESTs) that increased appeared to be novel. How some transcripts are selected in ischemic brain for translation into protein, while others are rejected, is not clear. The length of the 5′-UTR in the ischemically induced transcripts that occur in the NCBI RefSeq database did not indicate any general tendency to be more than 200 nt, nor to be longer than the 5′-UTRs of the unbound transcripts. Thus, the presence of a complex 5′-UTR region with internal ribosome entry sites (IRES) or polypyrimidine tracts (TOP) does not appear to be the basis of selection for translation in ischemic brain.

Permeability of the blood-brain barrier to mannitol in the rat following 2450 MHz microwave irradiation

The radiotracer method of Oldendorf was used to determine if 2450 MHz continuous wave (CW) microwave energy increases blood-brain barrier permeability to [14C]mannitol, which is normally excluded from entering the brain. Anesthetized, adult rats were irradiated singly for 30 min in the quiet zone of an anechoic chamber, at average power densities from 0.1 to 30 mW/sq.cm. Afterwards each rat received an intracarotid bolus injection of [14C]mannitol/[3H]water mixture and was decapitated 15 sec later. Uptake of [14C]mannitol relative to the highly permeable reference substance, [3H]water, was calculated as the brain uptake index (BUI) for 4 brain regions. Mean BUI values for tissues from the microwave-irradiated rats did not differ significantly from sham-irradiated animals, and a microwave influence on barrier permeability was not evident. Irrespective of treatment, BUI values for cerebellum and medulla were much higher and more variable than values for cortex or diencephalon, and were associated with reduced absorbance or retention of [3H]water. Because of a compromising influence of the vertebral arterial supply on the distribution of intracarotid-injected radiotracers, BUI measurements in caudal brain regions are probably unreliable unless accompanied by data on regional radioisotope concentrations. The absence of such data in an earlier BUI study, suggests that increases in BUI for cerebellum and medulla attributed to microwaves were possibly misinterpreted as differences in barrier permeability to [14C]saccharides, when in fact changes in blood flow and [3H]water influx or egress were responsible.

Evidence for pore-like opening of the blood-brain barrier following forebrain ischemia in rats

The nature of the delayed blood-brain barrier (BBB) opening that occurs in rats subjected to forebrain ischemia by the technique of two-vessel (carotid) occlusion plus hypovolemic hypotension (2VO ischemia) was probed by examining the simultaneous, trans-barrier movement of two hydrophilic, normally poorly permeative solutes of markedly different molecular size: sucrose (MW = 342) and inulin (MW approximately 5000). Pentobarbital-anesthetized, male, Sprague-Dawley rats (342-374 g) were subjected to 10 min of 2VO ischemia (tympanic temperature, 37.5-38.0 degrees C); 6 h later they were reanesthetized and, along with non-ischemic controls, injected i.v. with [14C]sucrose and [3H]inulin. Transfer constants (Kis) for blood-to-brain movement of the tracers and Vis (apparent initial volumes of tracer distribution) were determined for six brain regions by the multiple-time, graphical method (tracer circulation times from 3 to 30 min). Vis differed little or insignificantly between the two tracers, or between control and post-ischemic rats; the values did not suggest appreciable endothelial binding of either tracer that might lead to its uptake by adsorptive-phase endocytosis. In the controls, regional Kis +/- S.E.M. (nl g(-1) s(-1)) for inulin ranged from 0.18 +/- 0.04 to 0.31 +/- 0.09 and were significantly lower (P < 0.01) than Kis for sucrose (1.53 +/- 0.16-1.91 +/- 0.29). The Ki ratio (sucrose/inulin) across brain regions (mean, 6.6; S.E.M., 0.6) was much lower than would be expected according to the concept that movement of most organic non-electrolytes across the intact BBB occurs by dissolution in and diffusion through endothelial cell plasma membranes, at a rate proportional to the lipid solubility and diffusivity of the solute. This finding is interpreted as indicating that a portion of the transfer of sucrose and inulin occurred by a mechanism other than dissolution-diffusion (e.g., via pores or vesicles). In the post-ischemic rats, Kis for both tracers were elevated significantly (P < 0.01) in parietal cortex, striatum, hippocampus, and midbrain. The post-ischemic increases (delta Kis) in these regions were greater for sucrose (1.90-3.31 nl g(-1) s(-1)) than for inulin (0.80-1.33). Across brain regions the ratio between sucrose delta Ki and inulin delta Ki averaged 2.9 (S.E.M., 0.2), a value significantly greater than the ratio of 1 that would be expected were the BBB opening due to an enhancement of micropinocytosis and vesicular transport. The correspondence of the mean delta Ki ratio with the ratio of the free diffusion coefficients of the tracers (D(f, suc)/D(f, inu) = 2.9; water, 38 degrees C) suggests that the delayed opening of the BBB following 2VO ischemia involves the formation of trans- or paracellular, aqueous pores or channels.

Detection of Higher-Order 50- and 10-kbp DNA Fragments before Apoptotic Internucleosomal Cleavage after Transient Cerebral Ischemia:

DNA fragments of 50 and 10 kbp were found in ischemic brain in adult rats following two-vessel occlusion or in neonates following hypoxia–ischemia. These higher-order fragments were detected before any laddered oligonucleosomal DNA fragmentation characteristic of apoptosis. Both the 50- and 10-kbp fragments were also detected during necrosis produced by decapitation, but these led to smeared smaller fragments, not laddered patterns. End-group analysis showed the presence of both 3′-OH and 5′-OH ends in both the 50- and 10-kbp fragments but the predominance of 3′-OH ends in the laddered fragments. A higher proportion of 5′-OH to 3′-OH ends was found in the 10-kbp fragment compared to the larger 50-kbp fragment, suggesting a selective degradation of the 50-kbp DNA fragment to the laddered oligonucleosomal patterns. Overall, the mode of DNA fragmentation appeared different from that described in classic apoptosis of thymocytes.

A comparison of cathepsin B processing and distribution during neuronal death in rats following global ischemia or decapitation necrosis

The objective of this study was to examine the possible role of the cysteine protease cathepsin B (E.C. 3.4.22.1) in the delayed neuronal death in rats subjected to the two-vessel occlusion model of global ischemia. Immunohistochemistry of the hippocampus showed an alteration in the distribution of cathepsin B in CA1 neurons from a lysosomal pattern to a more intense label redistributed into the cytoplasm. This change was not detected until the neurons had become morphologically altered with obvious shrinkage of the cytoplasmic region. Western blotting and enzyme activity measurements of subcellular fractions, including lysosomes and a cell soluble fraction, demonstrated that there was an overall decrease in cathepsin B activity at this time but an increase in the proenzyme form, particularly in the soluble fraction. This was found to be completely different from the marked loss of all forms of cathepsin B in necrotic neurons following decapitation.

Post-ischemic hypothermia attenuates loss of the vascular basement membrane proteins, agrin and SPARC, and the blood–brain barrier disruption after global cerebral ischemia

Vascular basement membrane (BM) stabilizes brain vessels and inhibits endothelial cell cycle. Cerebral ischemia causes BM breakdown with the loss of structural BM components including collagens and laminins. In this study, the expression changes of the BM proteoglycan agrin, and the non-structural BM constituent SPARC (BM-40, osteonectin), were studied in brain vessels after global cerebral ischemia. A transient 20-min forebrain ischemia followed by 1, 6 or 24 h of reperfusion was induced in adult Sprague-Dawley rats by combined bilateral common carotid artery occlusion and hypotension (42-45 mm Hg). In a separate group of animals, a mild (32 degrees C) post-ischemic hypothermia was induced for 6 h, starting immediately after ischemia. RNA from approximately 500 brain vessels (20-100 microm) extracted by laser-capture microdissection (LCM) microscopy was used to determine the expression of proteoglycans agrin and SPARC mRNAs by quantitative PCR (Q-PCR). Protein expression was determined by immunohistochemistry in adjacent tissue sections. The BBB permeability was assessed using (3)H-sucrose as an in vivo tracer and by examining fibrinogen immunoreactivity in tissue sections. A transient global brain ischemia resulted in a significant (ANOVA, p<0.05; 6 animals/group) reduction in agrin and SPARC mRNAs in LCM-captured brain vessels 24 h after reperfusion. A time-dependent loss of agrin and SPARC from the BM during reperfusion was also observed by immunochemistry. A 6-h post-ischemic hypothermia reduced SPARC and agrin mRNA and protein losses, BBB transfer constant for (3)H-sucrose as well as fibrinogen extravasation 24 h after reperfusion. It is conluded that a transient post-ischemic hypothermia stabilizes brain vessels and reduces BBB disruption in part by preventing proteolytic degradation of regulatory BM constituents, SPARC and agrin.