Roman Gadamski

Poly(ADP-ribose) polymerase during reperfusion after transient forebrain ischemia: its role in brain edema and cell death.

The activation of poly(ADP-ribose) polymerase (PARP) in the reperfused brain after ischemia has been assumed but never has been directly presented. Our studies indicate a different dynamic of PARP activity alteration in hippocampus during reperfusion after 3 and 10 min of transient forebrain ischemia in gerbils. The phasic stimulation of PARP activity was observed during reperfusion 15 min, 120 min, and 4 d after 3 min of ischemia with subsequent lowering of its activity close to control value on the seventh day of reperfusion. After 10 min of ischemic insult, PARP activity significantly increased from the third to the seventh day of reperfusion. The protein level of PARP was not significantly changed during reperfusion after 3 and 10 min of ischemia, with one exception: On the third day after 10 min of ischemia, PARP protein level was 28% lower compared to control; however, no enhancement of 85-kDa protein immunoreactivity was observed. These data indicate the lack of PARP cleavage in hippocampus of gerbils subjected to ischemia-reperfusion injury. The inhibitor of PARP, 3-aminobenzamide (3-AB) in a dose of 30 mg/kg b.w. (body weight) injected intravenously directly after 3 min of ischemia protects >60% of neuronal cells against death in the CA1 layer of hippocampus but has no effect after 10 min of ischemic episode. 3-AB decreased forebrain edema significantly after 3 and 10 min of ischemia. Our data indicate that PARP inhibitor(s) might offer a potent therapeutic strategy for short global ischemia. The combination of PARP inhibitor with potent antioxidant might enhance its ameliorating effect.

Neuroprotective potential of group I metabotropic glutamate receptor antagonists in two ischemic models.

The neuroprotective potential of mGluR1 and mGluR5 antagonists (group I), EMQMCM and MTEP, respectively was studied using the 3 min forebrain ischemia model in Mongolian gerbils and the hypoxia-ischemia model in 7-day-old rats. Hypoxia-ischemia was induced by unilateral carotid occlusion followed by 75 min exposure to hypoxia (7.3% O(2) in N(2)), forebrain ischemia in gerbils was evoked by bilateral common carotid artery occlusion. The postischemic rectal body temperature in rat pups or brain temperature of gerbils was measured. The drugs were administered i.p. three times every 2 h after the insult, each time in equal doses of 1.25, 2.5 or 5.0 mg/kg. After 2 weeks brain damage was evaluated as weight decrease of the ipsilateral hemisphere in the rat pups or damage to CA1 pyramids in the gerbil hippocampus. The results demonstrated a dose dependent neuroprotection in both ischemic models by EMQMCM, while MTEP was neuroprotective only in the gerbil model of forebrain ischemia. EMQMCM reduced postischemic hyperthermia in gerbils. Thus, the antagonists of mGluR1 and mGluR5 show differential neuroprotective ability in two models of brain ischemia. Postischemic hypothermia may be partially involved in the mechanism of neuroprotection following EMQMCM in gerbils.

Changes in the NPY immunoreactivity in gerbil hippocampus after hypoxic and ischemic preconditioning

Preconditioning with sublethal ischemia or hypoxia may reduce the high susceptibility of CA1 pyramidal neurons to ischemic injury. In this study, we tested the hypothesis that enhanced level of neuropeptide Y (NPY) might play a role in the mechanisms responsible for this induced tolerance. Changes in NPY immunoreactivity in the hippocampal formation of preconditioned Mongolian gerbils were compared with the level of tolerance to test ischemia. Tolerance was induced by preconditioning with 2-min of ischemia or with three trials of mild hypobaric hypoxia (360 Torr, 2 h), separated by 24 h, that were completed 48 h before the 3-min test ischemia. The number of NPY-positive neurons in the gerbil hippocampal formation was assessed 2, 4 and 7 days after preconditioning. Survival of the CA1 pyramidal neurons was examined 14 days after the insult. Our experiments demonstrated that ischemic and hypoxic preconditioning produced equal attenuation of the damage evoked by 3-min ischemia, although the pattern of NPY immunoreactivity in the hippocampus differed. Preconditioning ischemia resulted in a 20% rise in the number of NPY-positive neurons 2 days later that disappeared 4 days after the ischemic episode, while mild hypobaric hypoxia induced a twofold increase in the number of NPY-positive neurons that lasted for at least 7 days. Although induced tolerance to ischemia 2 days after ischemic or hypoxic preconditioning was accompanied by increased immunoreactivity of NPY, there was no correlation between its intensity and the level of neuroprotection.

NMDA receptor antagonism does not inhibit induction of ischemic tolerance in gerbil brainin vivo

Effects of high and moderate affinity uncompetitive NMDA receptor antagonists (+)MK-801 and memantine on ischemic tolerance were compared in relation to telemetrically controlled brain temperature. The tolerance to an injurious 3 min test of global forebrain ischemia in Mongolian gerbils was induced 48 h earlier by 2 min preconditioning ischemia. Normothermic preconditioning was virtually harmless, and greatly reduced neurode-generation evoked by test ischemia. In hyperther-mic animals it was injurious and failed to induce tolerance. Memantine (5 mg/kg) and (+)MK-801 (3 mg/kg) injected i.p. 1 h before preconditioning did not inhibit ischemic tolerance in the normo-thermic gerbils, while in hyperthermic animals treated with (+)MK-801 ischemic tolerance was partially restored. Subchronic 3 day infusion of memantine (30 mg/kg/day) significantly decreased neurodegeneration, and preconditioning in the nor-mothermic gerbils further reduced neuronal damage. Hyperthermia exacerbated preconditioning ischemia and in this way reduced expression of tolerance, while (+)MK-801 partially reversed this effect. Our results do not confirm previous reports on the role of NMDA receptors in the induction of ischemic tolerance in gerbils.

Changes of the Na/K ATPase activity in the cerebral cortical microvessels of rat after single intraperitoneal administration of mercuric chloride : histochemical demonstration with light and electron microscopy

SummarySince inorganic mercury salts only poorly penetrate the cerebral microvascular endothelial cells comprising the blood-brain barrier (BBB), their neurotoxicity may be predicted to result from interference with BBB transport enzymes. In the present study, we tested the effect of mercuric chloride (HgCl2) on Na+/K+ ATPase activity, a key enzyme involved in the ion transport in and out of the brain. Routine histochemical staining in conjunction with light and electron microscopy was used to evaluate the changes in the Na+/K+ ATPase activity in cerebral cortical microvesels of rats who received a single intraperitoneal injection of 6 mg/kg HgCl2. At 1 h after HgCl2 administration, light microscopy revealed uniform reduction of the Na+/K+ ATPase reaction in all cortical layers. Electron microscopy confirmed the enzyme reaction to be very weak to completely absent in both the luminal and abluminal endothelial cell membranes, and the luminal plasmalemma showed invaginations and pinocytic vesicles indicative of changes in its transport functions. The enzyme inhibition coincided with, and was likely to contribute to, profound perivascular swelling, involving mainly the astrocytic endfeet. The enzyme activity showed a partial recovery 18 h after HgCl2 treatment, mainly in cortical layers II and III. After 5 days, the recovery of the enzyme activity appeared complete as observed by light and electron microscopy. The recovery of the microvascular Na+/K+ ATPase coincided with the appearance of a strongly positive Na+/K+ ATPase reaction in the adjacent astrocytic processes and with the diminution of perivascular swelling.

Protection against post-ischaemic neuronal loss in gerbil hippocampal CA1 by glycine b and AMPA antagonists

SummaryNovel antagonists of the glycineB site of the NMDA receptor (MRZ 2/570, MRZ 2/576), and an AMPA receptor antagonist, NBQX were tested in 3-min. global ischaemia in gerbils. Untreated animals showed after 14 days a loss of almost 90% of pyramidal neurones in the CA1 region, which was prevented by NBQX, and reduced to 50% by both glycineB antagonists. NBQX produced a delayed, long lasting (up to 24 hr) hypothermia while hypothermia with both glycineB antagonists was transient.

Retinal gliopathy accompanying thioacetamide-induced liver insufficiency: light and electron microscopic observations.

Abstract A recent examination of retinae of patients who had died with symptoms of liver insufficiency (LI) including hepatic encephalopathy (HE) revealed morphological changes in retinal Müller glia similar to the astrocytic changes normally accompanying HE, and the term “hepatic retinopathy” (HR) was coined to define these changes. In the present study, the immunomorphology and ultrastructure of Müller cells were examined in rats in which LI with accompanying HE was induced with a hepatotoxin, thioacetamide (TAA). Light microscopically, retinae of rats with LI were characterized by swelling of the Müller cell cytoplasm. Immunostaining for glia-specific marker proteins in Müller cells from LI rats revealed a strongly enhanced expression of glial fibrillary acidic protein, and a considerable increase in glutamine synthetase immunoreactivity, as compared to control animals. Ultrastructurally, the Müller cells of LI rats showed swelling and vacuolization of cell processes. In particular, the endfeet contained many swollen mitochondria. By contrast, LI produced no morphologically demonstrable changes in retinal neurons and photoreceptor cells. Thus, the retinal changes induced by TAA in the rats strongly resembled those described in human HR, rendering the present rat model suitable for more detailed investigations of the pathomechanism(s) of HR.

Antagonists of group I metabotropic glutamate receptors do not inhibit induction of ischemic tolerance in gerbil hippocampus

In this study we tested the effect of antagonists of two subtypes of the group I metabotropic glutamate receptors (mGluRs GI) on the induction of ischemic tolerance in relation to brain temperature. These experiments were prompted by indications that glutamate receptors may participate in the mechanisms of ischemic preconditioning. The role of NMDA receptors in the induction of ischemic tolerance has been debated while there is lack of information concerning the involvement of mGluRs GI in this phenomenon. The tolerance to injurious 3 min forebrain ischemia in Mongolian gerbils was induced 48 h earlier by 2 min preconditioning ischemia. Brain temperature was measured using telemetry equipment. EMQMCM and MTEP, antagonists of mGluR1 and mGluR5, respectively, were injected i.p. at a dose of 5 mg/kg. They were administered either before preconditioning ischemia in a single dose or after 2 min ischemia three times every 2 h. Both antagonists did not inhibit the induction of ischemic tolerance. Thus, our data indicate that group I metabotropic glutamate receptors do not play an essential role in the induction of ischemic tolerance.

Beneficial effects of GM1 ganglioside on photochemically-induced microvascular injury in cerebral cortex and hypophysis in rat

Gangliosides, the glycophospholipids which are abundantly present in the central nervous system, have been shown to stimulate neuronal regeneration and counteract the deleterious effects of ischemia on cerebral neurons. The further elucidate the mechanism of action of gangliosides in cerebral ischemia, we investigated the influence of GM1 ganglioside in the model of photochemically-induced microvascular injury in rat brain. The animals were injected with rose Bengal and illuminated through cranium with halogen lamp. This treatment resulted in the development of microthrombi and alterations in endothelial cells in the microvessels. Administration of 20 mg/kg GM1 ganglioside, 1 h before the photochemical reaction, largely reduced subsequent microvascular damage. In conclusion, the GM1 ganglioside is able to prevent microvascular damage in the central nervous system.

Differential effects of N-methyl-d-aspartate on Ca2+ homeostasis in developing and adult rat striatum: in vivo microdialysis approach

This in vivo study concerns developmental differences in the sensitivity of striatal neurons to N‐methyl‐d‐aspartate (NMDA). Changes in calcium homeostatis in adult vs immature rats at postnatal days 8–10, evoked by NMDA, were evaluated by measurements of 45Ca efflux and of Ca2+ taurine and phosphoethanolamine concentrations in striatal microdialysates. The efflux of [14C]sucrose was employed in order to measure changes in extracellular space volume. In adult rats the addition of 5 mM NMDA for 20 min to the perfusion medium resulted in a 30–40% increase in 45Ca efflux, and in a 15% increase in [14C]sucrose efflux. Ten minutes after NMDA perfusion, 45Ca and [14C]sucrose efflux returned to the baseline. No significant changes in Ca2+ or amino acid concentrations were observed in the dialysate of the adult rat striatum. NMDA perfusion in the striatum of immature rats initially induced a transient (5 min) increase in the efflux of 45Ca (by 13%) and [14C]sucrose (by 9%). This was followed by a prolonged (lasting 45–50 min) 45% decrease in 45Ca efflux, an 80% reduction of Ca2+ concentration, and increases in taurine and phosphoethanolamine concentrations in the dialysate, whereas [14C]sucrose efflux recovered within 10 min.