Sławomir Januszewski

Prolonged and concomitant induction of astroglial immunoreactivity of interleukin-1beta and interleukin-6 in the rat hippocampus after transient global ischemia.

We have investigated the pattern of expression of IL-1beta and IL-6 immunoreactivities in rat hippocampus after transient complete brain ischemia evoked by a 10 min cardiac arrest, at survival times ranging from 1 day to 28 days. To identify the cell types expressing the two immunoreactivities we used specific cell markers and combined staining procedures. In the intact brain IL-1beta and IL-6 were mainly localized in neurons particularly in pyramidal and granular cell layers. Ischemic insult resulted in a concomitant induction of IL-1beta and IL-6 immunoreactivities in multiple astroglia especially in the CA1 area which is the most vulnerable to ischemic insult as manifested by a massive delayed neuronal death accompanied by an intense gliosis. The number of astroglia expressing both immunoreactivities and the intensity of staining was maximal at the 14th day and remained at the same level at the 28th day. Our data suggest that the astroglial IL-1beta and IL-6 may affect the neurodegeneration of CA1 neurons in the ischemic hippocampus and that the prolonged proinflammatory effects of IL-1beta prevail over the presumed protective action of IL-6.

Sporadic Alzheimer’s Disease Begins as Episodes of Brain Ischemia and Ischemically Dysregulated Alzheimer’s Disease Genes

The study of sporadic Alzheimer’s disease etiology, now more than ever, needs an infusion of new concepts. Despite ongoing interest in Alzheimer’s disease, the basis of this entity is not yet clear. At present, the best-established and accepted “culprit” in Alzheimer’s disease pathology by most scientists is the amyloid, as the main molecular factor responsible for neurodegeneration in this disease. Abnormal upregulation of amyloid production or a disturbed clearance mechanism may lead to pathological accumulation of amyloid in brain according to the “amyloid hypothesis.” We will critically review these observations and highlight inconsistencies between the predictions of the “amyloid hypothesis” and the published data. There is still controversy over the role of amyloid in the pathological process. A question arises whether amyloid is responsible for the neurodegeneration or if it accumulates because of the neurodegeneration. Recent evidence suggests that the pathophysiology and neuropathology of Alzheimer’s disease comprises more than amyloid accumulation, tau protein pathology and finally brain atrophy with dementia. Nowadays, a handful of researchers share a newly emerged view that the ischemic episodes of brain best describe the pathogenic cascade, which eventually leads to neuronal loss, especially in hippocampus, with amyloid accumulation, tau protein pathology and irreversible dementia of Alzheimer type. The most persuasive evidences come from investigations of ischemically damaged brains of patients and from experimental ischemic brain studies that mimic Alzheimer-type dementia. This review attempts to depict what we know and do not know about the triggering factor of the Alzheimer’s disease, focusing on the possibility that the initial pathological trigger involves ischemic episodes and ischemia-induced gene dysregulation. The resulting brain ischemia dysregulates additionally expression of amyloid precursor protein and amyloid-processing enzyme genes that, in addition, ultimately compromise brain functions, leading over time to the complex alterations that characterize advanced sporadic Alzheimer’s disease. The identification of the genes involved in Alzheimer’s disease induced by ischemia will enable to further define the events leading to sporadic Alzheimer’s disease-related abnormalities. Additionally, knowledge gained from the above investigations should facilitate the elaboration of the effective treatment and/or prevention of Alzheimer’s disease.

Evidence of blood-brain barrier permeability/leakage for circulating human Alzheimer's β-amyloid (1-42)-peptide

BRAINS from patients with Alzheimers disease contain amyloid plaques which are composed of β-amyloid peptide and are considered to play a causal role in the neuropathology of this disease. The origin of β-amyloid peptide in brain parenchyma and vessels of Alzheimers disease patients is not known. This study examined the permeability of the blood-brain barrier to β-amyloid peptide in rats subjected to single or repeated episodes of global cerebral ischaemia followed by i.v. injections of human synthetic β-amyloid-(1–42)-peptide. Rats receiving β-amyloid peptide after ischaemia demonstrated multifocal and widespread accumulation of β-amyloid peptide in hippocampus, cerebral cortex and occasionally in white matter. β-Amyloid peptide penetration involved arterioles, veins and venules. Neuronal, glial and pericyte bodies were observed filled with β-amyloid peptide. Direct evidence that soluble human β-amyloid-(1–42)-peptide crosses the blood-brain barrier and enters the brain from the circulation is thus provided for the first time.

Concomitant up-regulation of astroglial high and low affinity nerve growth factor receptors in the CA1 hippocampal area following global transient cerebral ischemia in rat.

We have examined the effect of global transient cerebral ischemia, evoked in rat by 10 min of cardiac arrest, upon the changes in the cellular expression of two nerve growth factor (NGF) receptors (TrkA and p75) in the hippocampus. We have used immunocytochemical procedures, including a quantitative analysis of staining, along with some quantitative morphological analyses. We have found, under ischemic conditions, a decrease of TrkA immunoreactivity in degenerating CA1 pyramidal neurons and in neuropil. On the other hand, a strong, ischemia-induced up-regulation of TrkA and p75 immunoreactivity was observed in the majority of reactive astroglia population in the adjacent CA1 hippocampal region. The colocalization of the two receptors in the same reactive astroglial cells was evidenced by double immunostaining and further supported by quantitative morphological analysis of TrkA and p75 immunoreactive glial cells. Our data implicate the involvement of NGF receptors in the postischemic regulation of astrocytic function; however, the lack of NGF receptor expression on some astrocytes suggests heterogeneity of astroglia population. Our results also indicate that the lack of neuroprotective action of astroglial NGF induced in the ischemic hippocampus [J Neurosci Res 41 (1995) 684; Acta Neurobiol Exp 57 (1997) 31; Neuroscience 91 (1999) 1027] is not caused by a paucity of NGF receptors but may rather be due to the counteraction of some proinflammatory substances, released simultaneously by glia cells. On the other hand, the up-regulated astroglial TrkA receptor may be an important target for exogenous NGF, which, as previously described [J Neurosci 11 (1991) 2914; Neurosci Lett 141 (1992) 161], exerts a neuroprotective effect in ischemia.

Micro-blood-brain barrier openings and cytotoxic fragments of amyloid precursor protein accumulation in white matter after ischemic brain injury in long-lived rats.

Our study demonstrates that ischemia-reperfusion brain injury induces an increase in blood-brain barrier (BBB) permeability in the periventricular white matter. This chronic insufficiency of BBB may allow entry of neurotoxic fragments of amyloid precursor protein (APP) and other blood components such as platelets into the perineurovascular white matter tissue. These components may have secondary and chronic harmful effects on the ischemic myelin and axons and can intensify the phagocytic activity of microglial cells. Pathological accumulation of toxic fragments of APP in myelinated axons and oligodendrocytes appears after ischemic BBB injury and seem to be concomitant with, but independent of neuronal injury. It seems that ischemia-reperfusion disturbances may play important roles, both directly and indirectly, in the pathogenesis of white matter lesions. This pathology appears to have distribution similar to that of sporadic Alzheimers disease. We noted micro-BBB openings in ischemic white matter lesions that probably would act as seeds of future Alzheimers-type pathology.

Ischemic rats as a model in the study of the neurobiological role of human beta-amyloid peptide. Time-dependent disappearing diffuse amyloid plaques in brain.

Brains from patients with Alzheimers disease contain diffuse and senile amyloid plaques. Using an experimental model, we have addressed the issue whether diffuse plaques of amyloid persist, develop with time, or both, in rats injected with human beta-amyloid-(1-42)-peptide for 3 and 12 mon after brain ischemia. Rats receiving beta-amyloid peptide for 3 months after brain ischemia demonstrated widespread diffuse amyloid plaques in hippocampus and cerebral cortex. Neuronal, glial, ependymal, endothelial and pericyte cell bodies were observed filled with beta-amyloid peptide. No staining was observed in control brains. In the group alive 1 year no deposition of human beta-amyloid peptide was observed, too. Direct evidence that diffuse amyloid plaques can disappear in the brain is thus provided for the first time.

Increase of the brain uptake index for L-ornithine in rats with hepatic encephalopathy

We measured the brain uptake index (BUI) for radiolabelled L-ornithine (ORN) in rats with acute hepatic encephalopathy (HE) induced by two (onset stage) or three (comatous stage) administrations of a hepatotoxin-thioacetamide (TAA). In the comatose group, an increase of the BUI to 275% of control was measured at 24 h post-treatment. In the onset group, the BUI for ORN increased gradually with time: it reached 220% of control at 7 days post-treatment and 442% of control at 21 days post-treatment. HE did not raise the BUI for a blood-brain barrier (BBB) non-penetrable amino acid L-aspartate (ASP), indicating that HE activates ORN transport but does not produce BBB leakage. ORN transport through BBB was not increased in rats with hyperammonemia comparable to that accompanying HE, but was induced without liver damage. Considering recent evidence that ORN acting intracerebrally ameliorates pathophysiological symptoms of HE, increased transport ORN across BBB should facilitate HE therapy based on systemic administration of this amino acid.

Neuronal Autophagy: Self-eating or Self-cannibalism in Alzheimer’s Disease

Autophagy is a major intracellular degeneration pathway involved in the elimination and recycling of damaged organelles and long-lived proteins by lysosomes. Many of the pathological factors, which trigger neurodegenerative diseases, can perturb the autophagy activity, which is associated with misfolded protein aggregates accumulation in these disorders. Alzheimer’s disease, the first neurodegenerative disorder between dementias, is characterized by two aggregating proteins, β-amyloid peptide (plaques) and τ-protein (tangles). In Alzheimer’s disease autophagosomes dynamically form along neurites within neuronal cells and in synapses but effective clearance of these structures needs retrograde transportation towards the neuronal soma where there is a major concentration of lysosomes. Maturation of autophago-lysosomes and their retrograde trafficking are perturbed in Alzheimer’s disease, which causes a massive concentration of autophagy elements along degenerating neurites. Transportation system is disturbed along defected microtubules in Alzheimer’s disease brains. τ-protein has been found to control the stability of microtubules, however, phosphorylation of τ-protein or an increase in the total level of τ-protein can cause dysfunction of neuronal cells microtubules. Current evidence has shown that autophagy is developing in Alzheimer’s disease brains because of ineffective degradation of autophagosomes, which hold amyloid precursor protein-rich organelles and secretases important for β-amyloid peptides generation from amyloid precursor. The combination of raised autophagy induction and abnormal clearance of β-amyloid peptide-generating autophagic vacuoles creates circumstances helpful for β-amyloid peptide aggregation and accumulation in Alzheimer’s disease. However, the key role of autophagy in Alzheimer’s disease development is still under consideration today. One point of view suggests that abnormal autophagy induction causes a concentration of autophagic vacuoles rich in amyloid precursor protein, β-amyloid peptide and the elements crucial for its formation, whereas other hypothesis points to marred autophagic clearance or even decrease in autophagic effectiveness playing a role in maturation of Alzheimer’s disease. In this review we present the recent evidence linking autophagy to Alzheimer’s disease and the role of autophagic regulation in the development of full-blown Alzheimer’s disease.

Extracellular Concentrations of Taurine, Glutamate, and Aspartate in the Cerebral Cortex of Rats at the Asymptomatic Stage of Thioacetamide-Induced Hepatic Failure: Modulation by Ketamine Anesthesia

Subclinical hepatic encephalopathy (SHE) was produced in rats by two intraperitoneal injections of TAA at 24 h intervals and the animals were examined 21 days later. Concentrations of the neuroactive amino acids taurine (Tau), glutamate (Glu) and aspartate (Asp), were measured in the cerebral cortical microdialysates of thioacetamide (TAA)-treated and untreated control rats. During microdialysis some animals were awake while others were anesthetized with ketamine plus xylazine. There was no difference in the water content of cerebral cortical slices isolated from control and SHE rats, indicating a recovery from cerebral cortical edema that accompanies the acute, clinical phase of hepatic encephalopathy in this model. When microdialysis was carried out in awake rats, dialysate concentrations of all the three amino acids were 30% to 50% higher in SHE rats than in control rats. Ketamine anesthesia caused a 2.2% increase of water content of cerebral cortical slices and increased Asp, Glu, and Tau concentration in microdialysates of control rats. In SHE rats, ketamine anesthesia produced a similar degree of cerebral edema, however, it did not alter Asp and Glu concentrations in the microdialysates. These data may reflect on one hand a neuropathological process of excitotoxic neuronal damage related to increased Glu and Asp, on the other hand neuroprotection from neuronal swelling indicated by Tau redistribution in the cerebral cortex. The reduction of the effects of SHE on Glu and Asp content in ketamine-anesthesized rats is likely to be due to interference of ketamine with the NMDA receptor-mediated component of the SHE-evoked excitatory neurotransmitter efflux and/or reuptake of the two amino acids. By contrast, the SHE-related increase of Tau content was not affected by ketamine anesthesia, indicating that the mechanism(s) underlying SHE-evoked accumulation of Tau must be different from the mechanism causing release of excitatory amino acids. The results with ketamine advocate caution when using this anesthetic in studies employing the cerebral microdialysis technique for measurement of extracellular amino acids.

Possible Reverse Transport of β-Amyloid Peptide Across the Blood-Brain Barrier

Our experiments were performed to test the hypothesis that human β-amyloid peptide 42 (βA) is able to enter and exit the brain parenchyma through the blood-brain barrier. In an effort to determine the effect of βA in an animal model, we have injected βA i. v. into rats following single and repeated brain ischemia. Rats were sacrificed at 3 and 12 months after injection and βA was localized by monoclonal antibody (mAb) 4G8. The present observations revealed an abundant presence of βA in the extracellular space of the brain, which appeared to be dilated, and a vigorous uptake of βA into the cytoplasm of endothelial and ependymal cells, pericytes, astrocytes and neurons. Some of the βA deposits were associated and/or had migrated to the vessels and to the ventricles, and by 3 months a significant amount of βA was directly associated with the vessels and was observed inside the ventricular space. Virtually no soluble and aggregating βA was found in brain tissue 1 year later. This suggests that phagocytic pericytes and astrocytes take up exogenous βA in an attempt to clear the peptide from the brain extracellular space and deliver it to the circulation. Further, direct removal of βA from the ventricles by the bloodstream is also possible. These observations suggest that a reverse transport of βA across endothelial cells of mi-crovessels represents one of the possible mechanisms responsible for removal of extravasated βA. The findings of the present study indicate that in normal conditions βA is rapidly cleared from the cerebrospinal fluid and brain parenchyma, suggesting that irreversible changes in the physico-chemical properties of the cerebrovascular endothelial cell surface are involved in βA deposition in the brain in Alzheimer’s disease (AD).