Tadeusz Wieloch

Models for studying long-term recovery following forebrain ischemia in the rat. 2. A 2-vessel occlusion model

ABSTRACT— A model is described in which transient ischemia is induced in rats anaesthetized with N2O:O2 (70:30) by bilateral carotid artery clamping combined with a lowering of mean arterial blood pressure to 50 mm Hg, the latter being achieved by bleeding, or by bleeding supplemented with administration of trimetaphan or phentolamine. By the use of intubation, muscle paralysis with suxamethonium chloride, and insertion of tail arterial and venous catheters, it was possible to induce reversible ischemia for long‐term recovery studies.

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma.

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.

Calcium accumulation and neuronal damage in the rat hippocampus following cerebral ischemia.

The present study was undertaken to correlate calcium accumulation with the development of neuronal necrosis following transient ischemia. After 10 min of forebrain ischemia in the rat—a period that leads to reproducible damage of CA1 pyramidal cells—determination of calcium concentration and evaluation of morphological signs of cell body necrosis in the dorsal hippocampus were performed at various recirculation times. Tissue calcium concentration was not different from control at the end of ischemic period and did not change after 3, 6, 12, or 24 h of recirculation. However, after 48 h, calcium content increased significantly, with a further increase being seen after 72 h. At early recovery periods, only scattered necrotic neurons were observed. after 48 h, only 2 of 12 hemispheres showed more than 25 necrotic cells per section. More conspicuous neuronal death was observed after 72 h. The results thus demonstrate that net accumulation of calcium in regio superior of the hippocampus precedes marked necrosis of CA1 pyramidal cells. The results suggest that one primary event in the delayed death of these cells is membrane dysfunction with increased calcium cycling.

The distribution of hypoglycemic brain damage

SummaryRats were exposed to insulin-induced hypoglycemia resulting in periods of cerebral isoelectricity ranging from 10 to 60 min. After recovery with glucose, they were allowed to wake up and survive for 1 week. Control rats were recovered at the stage of EEG slowing. After sub-serial sectioning, the number and distribution of dying neurons was assessed in each brain region. Acid fuchsin was found to stain moribund neurons a brilliant red.Brains from control rats showed no dying neurons. From 10 to 60 min of cerebral isoelectricity, the number of dying neurons per brain correlated positively with the number of minutes of cerebral isoelectricity up to the maximum examined period of 60 min.Neuronal necrosis was found in the major brain regions vulnerable to several different insults. However, within each region the damage was not distributed as observed in ischemia.A superficial to deep gradient in the density of neuronal necrosis was seen in the cerebral cortex. More severe damage revealed a gradient in relation to the subjacent white matter as well. The caudatoputamen was involved more heavily near the white matter, and in more severely affected animals near the angle of the lateral ventricle. The hippocampus showed dense neuronal necrosis at the crest of the dentate gyrus and a gradient of increasing selective neuronal necrosis medially in CA1. The CA3 zone, while relatively resistant, showed neuronal necrosis in relation to the lateral ventricle in animals with hydrocephalus. Sharp demarcations between normal and damaged neuropil were found in the hippocampus. The periventricular amygdaloid nuclei showed damage closest to the lateral ventricles. The cerebellum was affected first near the foramina of Luschka, with damage occurring over the hemispheres in more severely affected animals. Purkinje cells were affected first, but basket cells were damaged as well. Rare necrotic neurons were seen in brain stem nuclei. The spinal cord showed necrosis of neurons in all areas of the gray matter. Infarction was not seen in this study.The possibility is discussed that a neurotoxic substance borne in the tissue fluid and cerebrospinal fluid (CSF) contributes to the pathogenesis of neuronal necrosis in hypoglycemic brain damage.

Postischemic Blockade of AMPA but Not NMDA Receptors Mitigates Neuronal Damage in the Rat Brain following Transient Severe Cerebral Ischemia

Glutamatergic transmission is an important factor in the development of neuronal death following transient cerebral ischemia. In this investigation the effects of N-methyl-d-aspartate (NMDA) and non-NMDA receptor antagonists on neuronal damage were studied in rats exposed to 10 min of transient cerebral ischemia induced by bilateral common carotid occlusion combined with hypotension. The animals were treated with a blocker of the ionotropic quisqualate or α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor, 2.3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), given postischemia as an intraperitoneal bolus dose of 30 mg kg−1 followed by an intravenous infusion of 75 μg min−1 for 6 h, or with the noncompetitive NMDA receptor blocker dizocilpine(MK-801) given 1 mg kg−1 i.p. at recirculation and 3 h postischemia, or with the competitive NMDA receptor antagonist dl-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 40116), 5 mg kg−1, given intraperitoneally at recirculation. Treatment with NBQX provided a significant reduction of neuronal damage in the hippocampal CA1 area by 44–69%, with the largest relative decrease in the temporal part of the hippocampus. In neocortex a significant decrease in the number of necrotic neurons was also noted. No protection could be seen following postischemic treatment with dizocilpine or CGP 40116. Our data demonstrate that AMPA but not NMDA receptor antagonists decrease neuronal damage following transient severe cerebral ischemia in the rat and that the protection by NBQX may be dependent on the severity of the ischemic insult. We propose that the AMPA receptor–mediated neurotoxicity could be due to ischemia-induced changes in the control mechanisms of AMPA receptor–coupled processes or to changes of AMPA receptor characteristics.

Plasma fibronectin supports neuronal survival and reduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis

Fibronectin performs essential roles in embryonic development and is prominently expressed during tissue repair. Two forms of fibronectin have been identified: plasma fibronectin (pFn), which is expressed by hepatocytes and secreted in soluble form into plasma; and cellular fibronectin (cFn), an insoluble form expressed locally by fibroblasts and other cell types and deposited and assembled into the extracellular matrix. To investigate the role of pFn in vivo, we generated pFn-deficient adult mice using Cre-loxP conditional gene-knockout technology. Here we show that pFn-deficient mice show increased neuronal apoptosis and larger infarction areas following transient focal cerebral ischemia. However, pFn is dispensable for skin-wound healing and hemostasis.

Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death

SummaryThe ultrastructural changes in the pyramidal neurons of the CA1 region of the hippocampus were studied 6 h, 24 h, 48 h, and 72 h following a transient 10 min period of cerebral ischemia induced by common carotid occlusion combined with hypotension. The pyramidal neurons showed delayed neuronal death (DND), i.e. at 24 h and 48 h postischemia few structural alterations were noted in the light microscope, while at 72 h extensive neuronal degeneration was apparent. The most prominent early ultrastructural changes were polysome disaggregation, and the appearance of electron-dense fluffy dark material associated with tubular saccules. Mitochondria and nuclear elements appeared intact until frank neuronal degeneration. The dark material accumulated with extended periods of recirculation in soma and in the main trunks of proximal dendrites, often beneath the plasma membrane, less frequently in the distal dendrites and seldom in spines. Protein synthesis inhibitors (anisomycin, cycloheximide) and an RNA synthesis inhibitor (actinomycin D), administered by intrahippocampal injections or subcutanously, did not mitigate neuronal damage. Therefore, DND is probably not apoptosis or a form of programmed cell death. We propose that the dark material accumulating in the postischemic period represents protein complexes, possibly aggregates of proteins or internalized plasma membrane fragments, which may disrupt vital cellular structure and functions, leading to cell death.

Influence of Acidosis on Lipid Peroxidation in Brain Tissues in vitro

To study the influence of acidosis on free radical formation and lipid peroxidation in brain tissues, homogenates fortified with ferrous ions and, in some experiments, with ascorbic acid were equilibrated with 5–15% O2 at pH values of 7.0, 6.5, 6.0, and 5.0, with subsequent measurements of thiobarbituric acid-reactive (TBAR) material, as well as of water- and lipid-soluble antioxidants (glutathione, ascorbate, and α-tocopherol) and phospholipid-bound fatty acids (FAs). Moderate to marked acidosis (pH 6.5–6.0) was found to grossly exaggerate the formation of TBAR material and the decrease in α-tocopherol content and to enhance degradation of phospholipid-bound, polyenoic FAs. These effects were reversed at pH 5.0, suggesting a pH optimum at pH 6.0–6.5. It is concluded that acidosis of a degree encountered in ischemic brain tissues has the potential of triggering increased free radical formation. This effect may involve increased formation of the protonated form of superoxide radicals, which is strongly prooxidant and lipid soluble, and/or the decompartmentalization of iron bound to cellular macromolecules like ferritin.

Neurochemical correlates to selective neuronal vulnerability.

Publisher Summary This chapter describes some of the special neurochemical features of the areas of the brain selectively vulnerable to ischemic and hypoglycemic insults. The chapter focuses on the neuronal connections to the vulnerable brain areas, on the distribution of receptors and transmitter content in the vulnerable areas, and on some current hypothesis of neuronal damage. Emphasis will be placed on a possible imbalance between excitation and inhibition of neurons as a factor in the development of neuronal necrosis, in particular the importance of excitatory transmitters, suggested to mediate ischemic and hypoglycemic brain damage. The amino acids glutamate and aspartate are major excitatory transmitters in the central neurons system. When present in high concentration they are neurotoxic and can play a role in the pathogenesis of several neurological diseases, such as temporal lobe epilepsy, Huntingtons disease, and olivopontocerebellar dystrophy.

Evidence for amelioration of ischaemic neuronal damage in the hippocampal formation by lesions of the perforant path.

The effect of lesions of two excitatory afferent pathways on the cellular damage in the hippocampus following complete cerebral ischaemia was investigated in the rat. Lesions transecting the perforant path led to a significant decrease in cellular damage in the hippocampal CA1 region ipsilateral to the lesion as compared to the contalateral side and to control. Lesions of the fimbria-fornix, on the other hand, had no significant effects. We propose that the protective effect of the perforant path lesions is due to removal of glutamatergic/aspartergic pathways and that release of these excitatory amino acids might be a critical factor for neuronal necrosis following cerebral ischaemia.