Thaddeus S. Nowak

Synthesis of a Stress Protein Following Transient Ischemia in the Gerbil

Abstract: In vitro translation products of gerbil brain preparations, obtained from animals killed during recirculation following transient ischemia, showed increased synthesis of a 70‐kilodalton stress protein, identified by two‐dimensional gel electrophoresis. Stimulation of stress protein synthesis was evident as early as 2 h after recirculation, at which time overall translation activity remained low. Expression of the 70‐kilodalton protein reached a maximum at 8 h recirculation, when incorporation into other translation products had returned to essentially control levels. Increased incorporation into the stress protein was still detectable after 24 h recirculation. Although the functional consequences of increased expression of this stress protein remain unknown, these results suggest that the gerbil ischemia model may provide a useful experimental system in which to study the involvement of this phenomenon in processes related to postischemic cell damage and recovery.

Changes in brain energy metabolism and protein synthesis following transient bilateral ischemia in the gerbil.

Abstract: The time course of the reduction in brain protein synthesis following transient bilateral ischemia in the gerbil was characterized and compared with changes in a number of metabolites related to brain energy metabolism. The recovery of brain protein synthesis was similar following ischemic periods of 5, 10, or 20 min; in vitro incorporation activity of brain supernatants was reduced to approximately 10% of control at 10 or 30 min recirculation, showed slight recovery at 60 min, and returned to 60% of control activity by 4 h. Protein synthesis activity was indistinguishable from control at 24 h. One minute of ischemia produced no detectable effect on protein synthesis measured after 30 min reperfusion; longer periods of ischemia resulted in progressive inhibition, with 5 min producing the maximal effect. Pentobarbital (50 mg/kg) increased by 1–2 min the threshold ischemic duration required to produce a given effect. Whereas most metabolites recovered quickly following 5 min ischemia, glycogen showed a delayed recovery comparable to that seen for protein synthesis. These results are discussed in relation to possible mechanisms for the coordinate regulation of brain energy metabolism and protein synthesis. An improved method for the fluorimetric measurement of guanine nucleotides is described.

Selective Glial Vulnerability following Transient Global Ischemia in Rat Brain

Global cerebral ischemia selectively damages neurons, but its contribution to glial cell death is uncertain. Accordingly, adult male. rats were sacrificed by perfusion fixation at 1, 2, 3, 5, and 14 days following 10 minutes of global ischemia. This insult produces CA1 hippocampal neuronal death at post-ischemic (PI) day 3, but minor or no damage to neurons in other regions. In situ end labeling (ISEL) and immunohistochemistry identified fragmented DNA of dead or dying glia and distinguished glial subtypes. Rare ISEL-positive oligodendroglia, astrocytes, and microglia were present in control brain. Apoptotic bodies and ISEL-positive glia significantly increased at PI day 1 in cortex and thalamus (p <0.05) but were similar to controls in other regions and at other PI intervals. Most were oligodendroglia, although ISEL-positive microglia and astrocytes were also observed. These results show that oligodendroglia die rapidly after brief global ischemia and are more sensitive than neurons in certain brain regions. Their selective vulnerability to ischemia may be responsible for the delayed white matter damage following anoxia or CO poisoning or that associated with white matter arteriopathies. Glial apoptosis could contribute to the DNA ladders of apoptotic oligonucleosomes that have been found in post-ischemic brain.

Mechanism of Delayed Increases in Kynurenine Pathway Metabolism in Damaged Brain Regions Following Transient Cerebral Ischemia

Abstract: Delayed increases in the levels of an endogenous N‐methyl‐D‐aspartate receptor agonist, quinolinic acid (QUIN), have been demonstrated following transient ischemia in the gerbil and were postulated to be secondary to induction of indoleamine‐2,3‐dioxygenase (IDO) and other enzymes of the L‐tryptophan‐kynurenine pathway. In the present study, proportional increases in IDO activity and QUIN concentrations were found 4 days after 10 min of cerebral ischemia, with both responses in hippocampus > striatum > cerebral cortex > thalamus. These increases paralleled the severity of local brain injury and inflammation. IDO activity and QUIN concentrations were unchanged in the cerebellum of postischemic gerbils, which is consistent with the preservation of blood flow and resultant absence of pathology in this region. Blood QUIN and L‐kynurenine concentrations were not affected by ischemia. Brain tissue QUIN levels at 4 days postischemia exceeded blood concentrations, minimizing a role for breakdown of the blood–brain barrier. Marked increases in the activity of kynureninase, kynurenine 3‐hydroxylase, and 3‐hydroxyanthranilate‐3,4‐dioxygenase were also detected in hippocampus but not in cerebellum on day 4 of recirculation. In vivo synthesis of [13C6]QUIN was demonstrated, using mass spectrometry, in hippocampus but not in cerebellum of 4‐day postischemic animals 1 h after intracisternal administration of L‐[13C6]tryptophan. However, accumulation of QUIN was demonstrated in both cerebellum and hippocampus of control gerbils following an intracisternal injection of 3‐hydroxyanthranilic acid, which verifies the availability of precursor to both regions when administered intracisternally. Notably, although IDO activity and QUIN concentrations were unchanged in the cerebellum of ischemic gerbils, both IDO activity and QUIN content were increased in cerebellum to approximately the same degree as in hippocampus, striatum, cerebral cortex, and thalamus 24 h after immune stimulation by systemic pokeweed mitogen administration, demonstrating that the cerebellum can increase IDO activity and QUIN content in response to immune activation. No changes in kynurenic acid concentrations in either hippocampus, cerebellum, or cerebrospinal fluid were observed in the postischemic gerbils compared with controls, in accordance with the unaffected activity of kynurenine aminotransferase activity. Collectively, these results support roles for IDO, kynureninase, kynurenine 3‐hydroxylase, and 3‐hydroxyanthranilate‐3,4‐dioxygenase in accelerating the conversion of L‐tryptophan and other substrates to QUIN in damaged brain regions following transient cerebral ischemia. Immunocytochemical results demonstrated the presence of macrophage infiltrates in hippocampus and other brain regions that parallel the extent of these biochemical changes. We hypothesize that increased kynurenine pathway metabolism after ischemia reflects the presence of macrophages and other reactive cell populations at sites of brain injury.

DNA fragmentation follows delayed neuronal death in CA1 neurons exposed to transient global ischemia in the rat

Apoptosis is an active, gene-directed process of cell death in which early fragmentation of nuclear DNA precedes morphological changes in the nucleus and, later, in the cytoplasm. In ischemia, biochemical studies have detected oligonucleosomes of apoptosis whereas sequential morphological studies show changes consistent with necrosis rather than apoptosis. To resolve this apparent discrepancy, we subjected rats to 10 minutes of transient forebrain ischemia followed by 1 to 14 days of reperfusion. Parameters evaluated in the CA1 region of the hippocampus included morphology, in situ end labeling (ISEL) of fragmented DNA, and expression of p53. Neurons were indistinguishable from controls at postischemic day 1 but displayed cytoplasmic basophilia or focal condensations at day 2; some neurons were slightly swollen and a few appeared normal. In situ end labeling was absent. At days 3 and 5, approximately 40 to 60% of CA1 neurons had shrunken eosinophilic cytoplasm and pyknotic nuclei, but only half of these were ISEL. By day 14, many of the necrotic neurons had been removed by phagocytes; those remaining retained mild ISEL. Neither p53 protein nor mRNA were identified in control or postischemic brain by in situ hybridization with riboprobes or by northern blot analysis. These results show that DNA fragmentation occurs after the development of delayed neuronal death in CA1 neurons subjected to 10 minutes of global ischemia. They suggest that mechanisms other than apoptosis may mediate the irreversible changes in the CA1 neurons in this model.

Global Cerebral Ischemia Associated with Cardiac Arrest in the Rat: I. Dynamics of Early Neuronal Changes

Light microscopic neuronal changes were studied in rats subjected to 10 min of global ischemia produced by compression of the major cardiac vessels. Observations of cresyl violet-stained sections revealed early changes involving predominantly GABAergic neurons in various locations. In rats killed 15 min after recirculation, the changes were characterized by the appearance of a clear peripheral zone with condensation of the remaining neuronal cytoplasm. After 1 h, these zones appeared to be compartmentalized into individual pearl-like vacuoles, especially prominent in the nucleus reticularis thalami. After 3 h, the cytoplasmic vacuoles disappeared and the neuronal changes, particularly in the cerebral cortex, striatum, hippocampus, and pars reticulata of the substantia nigra, consisted mainly of hyperchromasia or loss of Nissl substance. After 2 days, the cerebral cortex and thalamus contained occasional neurons with conspicuously large nucleoli. After 7 days, the hippocampus revealed an approximately 50% loss of CA1 pyramidal neurons, associated with intense microglial reactivity in the stratum radiatum, whereas the neuronal destruction was more complete in the nucleus reticularis thalami. Our observations suggest a possibility that early changes in GABAergic neurons may provide a period of neuronal disinhibition and thus contribute to an excitatory ischemic damage in regions connected by GABAergic circuitry.

The Heat Shock/Stress Response in Focal Cerebral Ischemia

Focal ischemia results in striking changes in gene expression. Induction of hsp72, a member of the family of 70 kDa heat shock/stress proteins is a widely studied component of the generalized cellular response to injury known as the ‘stress response’ that is detected in brain after ischemia and other insults. This overview summarizes observations on hsp72 expression in models of focal cerebral ischemia, considering its cellular distribution, factors affecting its transcriptional and translational expression, and its potential relevance to post‐ischemic pathophysiology. Hsp72 expression is essentially limited to regions in which cerebral blood flow falls below 50% of control levels, provided that residual perfusion allows synthesis of the induced mRNA and protein. The cellular distribution of hsp72 depends on the nature of the ischemic insult, with preferential vascular expression in severely ischemic territory that is destined to necrose, pronounced neuronal expression throughout the ischemic ‘penumbra’, and limited glial involvement in a narrow zone immediately surrounding the infarct. Together with results in other injury models, these observations indicate that hsp72 induction identifies discrete populations of surviving cells that are metabolically compromised, but not irreversibly damaged after focal ischemia. Available evidence suggests that the stress response is an important component of cellular defense mechanisms, and that successful accumulation of hsp72 is critical to survival following ischemia. Its expression may also contribute to mechanisms of induced ischemic tolerance. Future studies may be expected to more fully characterize the range of altered gene expression in response to focal ischemic injury and to establish specific roles for hsp72 and other induced proteins in the progression of injury and recovery following such insults

Kynurenine Pathway Enzymes in Brain: Responses to Ischemic Brain Injury Versus Systemic Immune Activation

Accumulation of l‐kynurenine and quinolinic acid (QUIN) in the brain occurs after either ischemic brain injury or after systemic administration of pokeweed mitogen. Although conversion of l‐[13C6]tryptophan to [13C6]‐QUIN has not been demonstrated in brain either from normal gerbils or from gerbils given pokeweed mitogen, direct conversion in brain tissue does occur 4 days after transient cerebral ischemia. Increased activities of enzymes distal to indoleamine‐2,3‐dioxygenase may determine whether l‐kynurenine is converted to QUIN. One day after 10 min of cerebral ischemia, the activities of kynureninase and 3‐hydroxy‐3,4‐dioxygenase were increased in the hippocampus, but local QUIN levels and the activities of the indoleamine‐2,3‐dioxygenase and kynurenine‐3‐hydroxylase were unchanged. By days 2 and 4 after ischemia, however, the activities of all of these enzymes in the hippocampus as well as QUIN levels were significantly increased. Kynurenine aminotransferase activity in the hippocampus was unchanged on days 1 and 2 after ischemia but was decreased on day 4, at a time when local kynurenic acid levels were unchanged. A putative precursor of QUIN, [13C6]anthranilic acid, was not converted to [13C6]‐QUIN in the hippocampus of either normal or 4‐day postischemic gerbils. Gerbil macrophages stimulated by endo‐toxin in vitro converted l‐[13C6]tryptophan to [13Ce]QUIN. Kinetic analysis of kynurenine‐3‐hydroxylase activity in the cerebral cortex of postischemic gerbils showed that Vmax increased, without changes in Km. Systemic administration of pokeweed mitogen increased indoleamine‐2,3‐dioxygenase and kynureninase activities in the brain without significant changes in kynurenine‐3‐hydroxylase or 3‐hydroxyanthranilate‐3,4‐dioxygenase activities. Increases in kynurenine‐3‐hydroxylase activity, in conjunction with induction of indoleamine‐2,3‐dioxygenase, kynureninase, and 3‐hydroxyanthranilate‐3,4‐dioxygenase in macro‐phage infiltrates at the site of brain injury, may explain the ability of postischemic hippocampus to convert l‐[13C6]tryptophan to [13C6]QUIN.

Delayed Increases in Regional Brain Quinolinic Acid Follow Transient Ischemia in the Gerbil

Excessive activity or release of excitatory amino acids has been implicated in the neuronal injury that follows transient cerebral ischemia. To investigate the metabolism of the endogenous excitotoxin, quinolinic acid, and its potential for mediating cell loss following ischemia, the concentrations of quinolinic acid, L-tryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid were quantified in gerbil brain regions at different times after 5 or 15 min of ischemia induced by bilateral carotid artery occlusion. Significant elevation of brain tryptophan levels, accompanied by increased 5-hydroxyindoleacetic acid concentrations, occurred during the first several hours of recirculation, but regional brain quinolinic acid concentrations were found either to decrease or remain unchanged during the first 24 h after the ischemic insult. However, significant increases in quinolinic acid concentrations occurred in striatum and hippocampus at 2 days of recirculation after 5 min of ischemia. After a further 4 and 7 days, strikingly large increases in quinolinic acid concentrations were observed in all regions examined, with the highest levels observed in the hippocampus and striatum, regions that also show the most severe ischemic injury. These delayed increases in brain quinolinic acid concentrations are suggested to reflect the presence of activated macrophages, reactive astrocytes, and/or microglia in vulnerable regions during and subsequent to ischemic injury. While the results do not support a role for increased quinolinic acid concentrations in early excitotoxic neuronal damage, the role of the delayed increases in brain quinolinic acid in the progression of postischemic injury and its relevance to postischemic brain function remain to be established.

Transient global ischemia in rats yields striatal projection neuron and interneuron loss resembling that in Huntington's disease

The various types of striatal projection neurons and interneurons show a differential pattern of loss in Huntingtons disease (HD). Since striatal injury has been suggested to involve similar mechanisms in transient global brain ischemia and HD, we examined the possibility that the patterns of survival for striatal neurons after transient global ischemic damage to the striatum in rats resemble that in HD. The perikarya of specific types of striatal interneurons were identified by histochemical or immunohistochemical labeling while projection neuron abundance was assessed by cresyl violet staining. Projectionneuron survival was assessed by neurotransmitter immunolabeling of their efferent fibers in striatal target areas. The relative survival of neuron types was determined quantitatively within the region of ischemic damage, and the degree of fiber loss in striatal target areas was quantified by computer-assisted image analysis. We found that NADPHd(+) and cholinergic interneurons were largely unaffected, even in the striatal area of maximal damage. Parvalbumin interneurons, however, were as vulnerable as projection neurons. Among immunolabeled striatal projection systems, striatoentopeduncular fibers survived global ischemia better than did striatopallidal or striatonigral fibers. The order of vulnerability observed in this study among the striatal projection systems, and the resistance to damage shown by NADPHd(+) and cholinergic interneurons, is similar to that reported in HD. The high vulnerability of projection neurons and parvalbumin interneurons to global ischemia also resembles that seen in HD. Our results thus indicate that global ischemic damage to striatum in rat closely mimics HD in its neuronal selectivity, which supports the notion that the mechanisms of injury may be similar in both.