Apoptosis induced in neuronal cultures by either the phosphatase inhibitor okadaic acid or the kinase inhibitor staurosporine is attenuated by isoquinolinesulfonamides H-7, H-8, and H-9.

Protein phosphorylation is kept in balance by an orchestrated action of kinases and phosphatases; when this balance is lost, neuronal apoptosis may occur. Okadaic acid (OKA), a marine toxin that inhibits specifically protein phosphatases 1 and 2A (EC 3.1.3.16), and staurosporine, an inhibitor of protein kinase C (PKC; EC 2.7.1.37), induced apoptosis in primary cultures of rat cerebellar granule neurons. We assayed apoptosis by the DNA gel electrophoresis, by the in situ TUNEL assay, and by morphological appearance following propidium iodide staining. Cell viability was assessed by the Trypan blue assay. Both OKA- and staurosporine-induced neuronal apoptosis were prevented by a macromolecular synthesis inhibitor actinomycin D and by a group of isoquinolinesulfonamide kinase inhibitors (H-7, 1-[5-isoquinolinesulfonyl]-2-methylpiperazine; H-8, N-¿2-[methylamino]ethyl¿-5-isoquinolinesulfonamide; H-9, N-(2-aminoethyl)-5-isoquinolinesulfonamide, but not by inhibitors of PKC, cyclic-GMP- and cyclic-AMP-dependent kinases, calcium/calmodulin-dependent kinases, tyrosine kinases, or by antioxidants. We postulate that a common mechanism, possibly an increased protein phosphorylation, is responsible for apoptosis triggered by an inhibition of phosphatases 1 and 2A and PKC. Elucidating the isoquinolinesulfonamide-sensitive mechanism may help us find new therapies for neurodegenerative diseases that involve apoptosis.

GABA and NMDA in the prevention of apoptotic-like cell death in vitro.

We have shown recently that cerebellar granule neurons die in the absence of depolarizing concentrations of KCl through an apoptosis-like process. To study the contributions of inhibitory (gamma-aminobutyric acid; GABA) and excitatory (glutamate) neurotransmitters in the prevention of apoptotic-like cell death in cultures grown in the presence of reduced concentrations of KCl (12.5 mM), we treated these cultures either acutely or chronically with GABA, bicuculline methiodide, a GABAA receptor antagonist, N-methyl-D-aspartate (NMDA) and/or the NMDA receptor antagonist, MK-801. Cell viability was measured with fluorescein diacetate/propidium iodide (FDA/PI) and trypan blue exclusion tests. In addition, DNA fragmentation was assessed quantitatively using an in situ terminal deoxynucleotidyl transferase assay. Our results demonstrate that treatment of cerebellar granule cell cultures maintained in 12.5 mM KCl with the glutamate receptor agonist NMDA and/or bicuculline protects against cell death and reduces DNA fragmentation. In contrast, GABA potentiated cerebellar granule cell apoptosis mediated by KCl deprivation. These data indicate that signal transduction pathways activated following NMDA receptor stimulation mimic the anti-apoptotic action of high potassium in primary cultures of cerebellar granule neurons. Also, our data support an inhibitory (hyperpolarizing) role for GABA in these cultures. Collectively, the results suggest that the neurotrophic actions of NMDA on granule cells maintained in low KCl and GABA on granule cells cultured in high KCl are due to the necessity for maintaining appropriate intraneuronal calcium concentrations.

Age-dependent increase in infarct volume following photochemically induced cerebral infarction: putative role of astroglia.

This study demonstrates that the photochemically induced model of stroke is an extremely viable method of inducing cerebral infarction in old animals. The lesions are reproducible both in terms of location and size and compatible with long-term survival of the animal. With this model we demonstrated, one week following surgery, a significantly larger infarct in rats 20 and 24 months of age compared to 4-month-old rats. The older rats also sustained greater neurologic deficits as assessed on a rotarod task. Older rats also were characterized by a glial response that was far less intense than in young animals. While the precise relationship between glia activation and cerebral damage remains to be determined, it would appear that a better understanding of those factors that contribute to the astrocytic response in the aged rat may be of particular benefit in designing therapeutic strategies aimed at reducing the pathologic consequences of cerebral infarction in elderly humans.

Electrobehavioral characteristics of epileptic rats following photothrombotic brain infarction.

The goal of this study was to characterize the electroencephalographic (EEG) and behavioral properties of young adult rats during extended video-EEG monitoring following photothrombotic brain infarction. Two-month-old male Sprague-Dawley rats underwent photothrombotic brain infarction of the left sensorimotor cortex with the photosensitive dye rose bengal (n=10) or were used as controls (n=9). Qualitative and quantitative EEG analysis was performed on digital video-EEG records obtained during 6 months of recording. The main finding of this study was that 5/10 (50%) lesioned animals developed focal epileptic seizures ipsilateral to the cortical infarct characterized by rhythmic spike-wave discharges with or without behavioral change. Epileptic animals demonstrated increased delta, theta, and low beta-range power ipsilateral to the infarct that reliably distinguished them from lesioned nonepileptic and control animals. Lesioned animals (epileptic and nonepileptic) also demonstrated a distinct pattern of focal rhythmic theta activity before or after generalized high beta-range discharges. Electrical and behavioral characteristics common to both lesioned and control animals included: (1) focal rhythmic theta activity in either hemisphere; (2) focal low beta-range discharges in either hemisphere; (3) generalized high beta-range discharges; (4) absence seizures; (5) generalized pseudoperiodic spike discharges associated with mild multifocal body jerks; (6) tonic-clonic seizures (one nai;ve control; one lesioned animal). Cresyl violet staining of lesioned animals' brains showed consistent infarcts of the sensorimotor cortex extending to the subcortical white matter. These results provide an expanded electrobehavioral description of young adult rats following photothrombotic brain infarction and augment further investigation into the molecular, cellular, and network alterations that contribute to the establishment of post-stroke epilepsy.

[Sources of the vascularization of the human interatrial septum of the heart with different variants of the atrial blood supply]. / Istochniki vaskuliarizatsii mezhpredserdnoi peregorodki serdtsa cheloveka pri razlichnykh variantakh krovosnabzheniia predserdii.

Angiograms of 110 preparations of the human hearts at various ages have been studied. In order to reveal vessels of the coronary bed, roentgen contrast lead mass has been used. Peculiarities of vascularization have been described in all parts of the interatrial septum and in the fossa ovalis area at various variants of branching of the atrial vessels: right coronary, left coronary and even. With age, in the fossa ovalis area certain decrease in the number of the blood vessels is observed and the network they form becomes less dense. In the interatrial septum its middle part and the fossa ovalis area are the regions which contain the least amount of vessels. In the antero-inferior part of this septum a comparatively dense vascular network is revealed.

[Sources of vascularization of the interventricular septum and septal papillary muscles in various types of blood supply in humans]. / Istochniki vaskuliarizatsii mezhzheludochkovoi peregorodki i peregorodochnykh sosochkovykh myshts serdtsa pri razlichnykh tipakh ego krovosnabzheniia u cheloveka.

Using the preparation method, roentgenoangiography by means of contrast lead mass and polychromic injection of the coronary vessels, vascularization of the interventricular septum (IVS) and the septal papillary muscles (SPM) has been studied on 49 preparations of human hearts (age 19-89) The data are presented on distribution pattern of the IVS and SPM of the coronary arteries branches at various types of the cardiac blood supply. Angioarchitectonics of the IVS is described along its whole length. Topographic peculiarities of the SPM blood supply on dependence of their position on the IVS are noted.

Opposite effect of protein synthesis inhibitors on potassium deficiency-induced apoptotic cell death in immature and mature neuronal cultures.

Typically, primary cultures of rat cerebellar granule neurons are grown in the presence of 25 mM KCl and are considered to mature by approximately 7 days in vitro. Potassium deficiency was created by growing the neurons from days 1 to 4 in the presence of 12.5 mM KCl (immature cultures) or by switching the mature neurons grown with 25 mM KCl to 12.5 mM KCl. In both conditions we observed neuronal death that bears the signs of apoptosis, i.e., DNA fragmentation determined qualitatively by agarose gel electrophoresis of DNA and quantitatively by in situ terminal deoxynucleotidyl transferase assay. The protein synthesis inhibitors cycloheximide and anisomycin provided neuroprotection in the mature cultures but potentiated the toxic effect of KCl deprivation in the immature neurons. The results suggest that a prudent use of protein synthesis inhibitors is critical in experiments with primary neuronal cultures.

Characterization of zinc-induced neuronal death in primary cultures of rat cerebellar granule cells.

Although zinc is essential for the activity of numerous biological systems, and zinc deficiency has been associated with various pathologies, this metal can also exert direct neurotoxic action. In primary cultures of rat cerebellar granule neurons, a brief, 15- to 30-min exposure to zinc (100-500 microM) resulted in concentration-dependent delayed neuronal death. The toxicity of zinc depended on the maturity of the neuronal cultures-it was not apparent prior to Day 5 and it reached a plateau at about 9-10 days in vitro. We assayed cell injury by measuring mitochondrial functioning (MTT assay) and cell death with the trypan blue exclusion assay. Apoptosis was assayed by the morphological appearance of cells following fluorescence staining with propidium iodide and by the in situ TUNEL technique. Mitochondrial injury was an early result of zinc treatment. Actinomycin D, an inhibitor of macromolecular synthesis, attenuated delayed cell death. The calcium channel blockers nimodipine and amlodipine reduced both mitochondrial injury and cell death; the blockade of ionotropic glutamate receptors with MK-801 or CNQX was ineffective. These results suggest that calcium channel-blocker-sensitive mitochondrial injury and DNA damage are operative in the protein-synthesis-dependent neurotoxicity of zinc. An imbalance of zinc homeostasis might play a role in the pathophysiology of apoptosis-associated neurodegenerative disorders.

Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: protection with antioxidants and glutamate receptor antagonists.

Cerebellar granule neurons undergo apoptosis when deprived of chronic depolarization; serum deprivation has not been considered as a trigger of apoptosis in this culture. Here we report that serum removal triggers cell injury, which is characterized by signs of apoptosis. Actual cell death (trypan blue permeability) occurred 24 and 48 hr after serum removal. At earlier times (6 and 8 hr after serum removal) we found significant impairment of mitochondrial functioning [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay] and an increase in the percentage of neurons showing signs of DNA fragmentation (insitu terminal deoxynucleotidyl transferase assay, fluorescent assay). Protection was obtained by inhibiting RNA synthesis with actinomycin D and by antioxidants [1mM: 1,4-diazobicyclo(2.2.2)octane, histidine, mannitol; 1% dimethyl sulfoxide; 0.01-1 microM ascorbic acid]. We also measured neuronal oxidation utilizing the oxidation-sensitive fluorescent dye 2', 7'-dichloro- fluorescin diacetate, and found a significant increase in the rate of neuronal oxidation as early as 15 min after serum deprivation. The blockade of glutamate receptors by (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione also provided neuroprotection. However, oxidative stress appears to precede glutamate receptor activation: within the 8 hr period of serum deprivation, mannitol was protective when present either during only the first or last 4 hr; MK-801 was protective only when present for the entire 8 hr period or in the last, but not first 4 hr of serum deprivation. Serum deprivation of mature cerebellar granule neurons can be used to study mechanisms of oxidative stress-induced apoptosis.

[Age-related characteristics of intraorganic vascular structures of the cerebral cortex, heart, kidneys and spleen in rats in the normal state and in arterial hypertension]. / Vozrastnye osobennosti vnutriorgannoi angioarkhitektoniki kory bol'shogo mozga, serdtsa, pochek i selezenki u krys v norme i pri arterial'noi gipertenzii.

The work has been performed on 40 normotensive and spontaneously hypertensive rats at the age of 6 months-1 year. In all the organs studied a direct dependence between the degree of changes in the intraorganic arterial walls and a relative content of the arterial vessels in the organ tissues has been revealed. The functional changes of the arterial bed of the organs mentioned in spontaneously hypertensive rats during hypertensive phase at the stage of stable hypertension, as a result of a prolonged adaptation transform into organic ones.

Neuronal apoptosis in an in vitro model of photochemically induced oxidative stress.

In neurons, oxidative stress can be triggered by neurotransmitter-linked mechanisms and may lead to apoptotic cell death. A simple and reproducible model of inducing oxidative stress is needed to elucidate mechanisms which link oxidative stress and neuronal apoptosis. We report here a method of inducing apoptosis in cell cultures by loading them with a photosensitive dye, rose bengal, and exposing the cultures to light, a procedure which generates reactive singlet oxygen. We used this model in primary culture of rat cerebellar granule neurons, and in a nonneuronal human embryonic kidney 293 cell line. We have measured the following: (a) metabolic activity of the mitochondria by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), (b) DNA fragmentation by quantitative in situ terminal deoxynucleotidyl transferase assay, and (c) cell viability by a trypan blue exclusion test. The oxidative stress caused an early impairment of mitochondrial function (MTT assay). This was followed by DNA fragmentation and ultimately by cell death. Protection was obtained with an inhibitor of macromolecular synthesis, anisomycin, and with antioxidant, vitamin E. This model can be used to study the mechanism of oxidative stress-triggered neuronal apoptosis, and it may help in discovering new targets for neuroprotective drugs.

[Age-related characteristics of the organic angioarchitectonics of normal rats and in arterial hypertension]. / Vozrastnye osobennosti angioarkhitektoniki nekotorykh organov krys v norme i pri arterial'noi gipertenzii.

Architectonics of intraorganic arterial vessels in the cervical cortex, heart, kidneys and spleen have been investigated in 40 normotensive and spontaneously hypertensive rats, 6 months and 1 year old. In all the organs studied a direct dependence has been revealed between the degree of changes in the intraorganic arteries and relative content of the arterial vessels in the tissue organs. Functional changes of the arterial vessels in the organs studied observed during the hypertensive phase, transfer into the organic ones, as a result of prolonged adaptation, when the stage of a stable hypertension takes place.