Effect of oral administration of pig spinal cord hydrolysate on clinical and histopathological symptoms of experimental allergic encephalomyelitis in rats.

Oral tolerance is the natural occurring phenomenon of a decreased immune response to previously fed antigens, which prevents induction of a response to dietary antigens. One of the mechanisms is deletion of T lymphocytes reactive to the fed antigen. Knowing that phenomenon, it seems appropriate to engage this mechanism for treatment of autoimmune diseases. Multiple sclerosis (MS) is an autoimmunological disease which causes neurological impairment in humans. Autoreactive T lymphocytes migrate through the open blood-brain barrier into the central nervous system (CNS), where they recognize myelin antigens as foreign, and induce an inflammatory response against the myelin sheath, which causes demyelination and even axonal loss. Experimental allergic encephalomyelitis (EAE), an animal model of MS, resembles the autoimmunological aspect of the disease. We used a broad spectrum of myelin antigens to induce EAE, and also to induce oral tolerance by giving myelin epitopes intragastrically to rats. The aim of our study was to evaluate whether pig spinal cord hydrolysate given intragastrically is able to evoke oral tolerance in rats with an animal model of MS - EAE. In our experiments we fed female Lewis rats with pig spinal cord hydrolysate at doses of 5, 20 and 100 mg per kg of body weight. We observed diminished clinical symptoms of ongoing EAE in rats fed with all doses of pig spinal cord hydrolysate. In the histopathological study, intensity of the inflammatory process in spinal cord was similar in rats not fed with EAE and in rats fed with lower doses of pig spinal cord hydrolysate. In animals fed with the highest dose of pig spinal cord hydrolysate, intensification of the inflammatory response was observed. These results were confirmed by morphometric evaluations. We found that feeding animals with preparations containing myelin antigens can reduce EAE symptoms, which may indicate oral tolerance induction, but the obtained results also underline the importance of dose of the orally given antigens, because of the possibility of enhancement of the inflammatory process in the CNS.

Diverse expression of selected SMN complex proteins in humans with sporadic amyotrophic lateral sclerosis and in a transgenic rat model of familial form of the disease.

BACKGROUND AND OBJECTIVE: There is circumstantial evidence linking sporadic amyotrophic lateral sclerosis (ALS) cases to a malfunction or deficit of a multimeric SMN complex that scrutinizes cellular RNAs; the core of this complex is survival motor neuron (SMN, or gemin 1) protein. We intended to verify this hypothesis by comparing the expression of both SMN and several other functionally associated gemins in the anterior horn motoneurons of patients who died of sporadic ALS (sALS), of transgenic rats with overexpression of the mutated human superoxide dismutase 1 gene (SOD1(G93A)) that represent a model of familial ALS (fALS), and of the respective controls. METHODS: Using archival material of paraffin blocks with samples of human and rat spinal cords, immunohistochemical reactions with antibodies against SMN and gemins 2, 3, and 4 were performed and assessed by light microscopy. RESULTS: The expression of SMN and all other studied gemins was observed in motoneurons of sALS patients, fALS rats, and in all controls, although the intensity varied. The immunolabeling was most intense in sALS patients with relatively fast disease course, and decreased with increasing disease duration in both the human sALS and rat fALS material. Irrespective of the disease stage, sALS material showed no or very low gemin 2 immunoreactivity, while clear gemin 2 immunoreactivity was observed in all fALS rats and control material. CONCLUSION: The deficient expression of gemin 2 in spinal cord motoneurons in human sALS may lead to a dysfunction and loss of neuroprotective action of the SMN complex.

Non-woven nanofiber mats - a new perspective for experimental studies of the central nervous system?

(Sub)chronic local drug application is clearly superior to systemic administration, but may be associated with substantial obstacles, particularly regarding the applications to highly sensitive central nervous system (CNS) structures that are shielded from the outer environment by the blood-brain barrier. Violation of the integrity of the barrier and CNS tissues by a permanently implanted probe or cannula meant for prolonged administration of drugs into specific CNS structures can be a severe confounding factor because of the resulting inflammatory reactions. In this study, we tested the utility of a novel way for (sub)chronic local delivery of highly active (i.e., used in very low amounts) drugs to the rat spinal cord employing a non-woven nanofiber mat dressing. To this end, we compared the morphology and motoneuron ( + ) counts in spinal cord cervical and lumbar segments between rats with glutamate-loaded nanofiber mats applied to the lumbar enlargement and rats with analogical implants carrying no glutamate. Half of the rats with glutamate-loaded implants were given daily valproate treatment to test its potential for counteracting the detrimental effects of glutamate excess. The mats were prepared in-house by electrospinning of an emulsion made of a solution of the biocompatible and biodegradable poly(L-lactide-co-caprolactone) polymer in a mixture of organic solvents, an aqueous phase with or without monosodium glutamate, and sodium dodecyl sulfate as an emulsifier; the final glutamate content was 1.4 µg/mg of the mat. Three weeks after mat implantation there was no inflammation or considerable damage of the spinal cord motoneuron population in the rats with the subarachnoid dressing of a glutamate-free mat, whereas the spinal cords of the rats with glutamate-loaded nanofiber mats showed clear symptoms of excitotoxic damage and a substantial increase in dying/damaged motoneuron numbers in both segments studied. The rats given systemic valproate treatment showed significantly lower percentages of damaged/dying motoneurons in their lumbar enlargements. These results demonstrate the capacity of nanofiber mats for generation of neurotoxic glutamate in the rat CNS. However, the tested nanofiber mats need further improvements aimed at extending the period of effective drug release and rendering the release more steady.

Immunoexpression of gemins 2 and 4 in the rat spinal cord. Is the SMN complex a new target in investigations of sporadic amyotrophic lateral sclerosis pathogenesis?

Sporadic amyotrophic lateral sclerosis (sALS) is a neurodegenerative disease leading to degeneration and loss of motoneurons in different structures of the nervous system. Although aetiology of the disease is unknown, it is hypothesized that the survival motor neuron (SMN) protein which protects motoneurons in spinal muscular atrophy, may play a similar role in ALS. Relatively little is known about normal expression and functions of the SMN complex compounds, i.e. SMN protein and the related gemins. Therefore, we have decided to examine the physiological expression of SMN and gemins 2 and 4 in spinal cords of healthy Wistar rats at different age using immunofluorescence and immunohistochemical methods. Our study revealed that (1) in rat spinal cord neurons, the immunoexpression of SMN and gemins 2 and 4 is present through the whole animal lifespan although the reactive cells reveal different intensity of the immunolabeling, (2) both SMN and gemin 2, and SMN and gemin 4 are present in the same motoneurons, (3) immunoexpression of gemin 2 and 4 decreases slightly with aging.

Survival motor neuron: motor neuron insurance for a whole lifespan?

The SMN (survival motor neuron) gene plays an important role in ontogenesis and its dysfunction leads to immatu-rity of skeletal muscles and motor neurons in the spinal cord. As a result of SMN mutations the affected cells die and clinical symptoms of spinal muscular atrophy (SMA) develop. Physiologically, SMN together with gemins is part of a multiprotein complex of particular importance to motor neuron development. Since the SMN gene is necessary for normal motor neuron maturity, a question arises whether its expression is preserved in postnatal life or finishes with the end of ontogenesis. To answer this question we examined expression of SMN and gemins 2, 3 and 4 in spinal cords of Wistar rats at age 1-350 days using immunofluorescence and immunohistochemical methods. In the examined animals expression of SMN appeared in neurons in 20-day old rats and increased with animal age. In rats aged 30-350 days SMN immunoreactivity was similar in all the examined animals. The same phenomenon was observed in assessment of gemin expression. Our study revealed that in rat spinal cord expression of SMN and gemins 2, 3 and 4 is present through a whole animal lifespan and not only in motor but also in sensory and autonomic neurons.

Is the spinal cord motoneuron exclusively a target in ALS? Comparison between astroglial reactivity in a rat model of familial ALS and in human sporadic ALS cases.

OBJECTIVE: Motoneurons are the focus of most investigations of amyotrophic lateral sclerosis (ALS), while the astrocyte reaction is regarded as a phenomenon secondary to neuron degeneration. Since astroglial reactivity differed in different studies of human and animal ALS models and often varied from case to case, we examined and compared astrocyte reactivity within the anterior horns of the spinal cord in a transgenic rat model of familial ALS and in human sporadic ALS (sALS) cases. METHODS: Routine histological staining and immunohistochemical reactions to cytoskeletal proteins [neurofilaments, glial fibrillary acidic protein (GFAP), vimentin and tau] and proliferative markers (proliferating cell nuclear antigen and Ki-67). RESULTS: In human sALS cases and in rats at the early pre-symptomatic and symptomatic stages of the disease, the astroglial reaction was very weak, although there was visible evidence of the morphological manifestations of motoneuron degeneration. Poor immunoreactivity to the GFAP and vimentin antigens and increased expression of tau protein were observed in astrocytes, particularly in the rat model. The astrocyte reaction was evident during a short 'transient' phase of the disease in animals, between the asymptomatic and paretic stages. Proliferating cell nuclear antigen immunoreactivity in glial and neuronal nuclei was observed only in animal material. CONCLUSIONS: Abnormalities in astrocyte cytoskeletal proteins are characteristic features for ALS, both in acquired and congenital forms of the disease. The cytoskeletal aberrations may lead to astroglial dysfunction and disturbances in glutamate uptake that may in turn increase the degeneration of motoneurons.

Unexpected morphological changes within hippocampal structures in a photochemical ring model of cerebral ischaemia.

A photochemical ring model of ischaemia was introduced in the middle of the nineteen eighties. Irradiation by a laser or arc lamp followed by intravenous injection of rose bengal resulted in thrombosis of pial and superficial cortical vessels. This ring model imitated focal ischaemic damage in humans. In our experiment twenty-seven Wistar rats of both sexes weighing 250-300 grams were examined. A photochemical ring model based on irradiation of the area of parietal bone 4 mm posteriorly to the bregma and 4 mm laterally from the sagittal suture was applied. A ring-shaped light beam with a wavelength of 510-540 nm with 5 mm diameter was generated by a high pressure discharge lamp at a power of 400 W. Two groups of rats treated and untreated with MK-801 and two rings of the thickness of 0.35 mm and 0.5 mm were used in the experiment. Morphological examination was performed in animals sacrificed 1 and 4 days after the irradiation. On formalin-fixed and paraffin-embedded slices HE staining method and immunoreaction with antibodies to ubiquitin were applied. Our material confirmed well known information about the dynamics of infarct breakdown, ischaemic-induced angiogenesis, glial reaction and other typical changes described previously in handbooks and numerous papers. In the experiment, morphological changes were more intensive after the irradiation by 0.5 mm than 0.35 mm irradiating rings and 4 days than one day after the irradiation. A surprising finding observed in some of the examined animals was more intensive neuronal damage after treatment with MK-801. Another unpredicted discovery was intensive morphological alterations found in CA4 and CA3 hippocampal sectors. Moreover, these alterations were not limited to the damaged hemisphere, but were also observed contralaterally. In some of the rats, ischaemic and necrotic cells were additionally found within both parasagittal areas. We connect this atypical localization of the ischaemic changes with dispersion of light emitted by the used lamp. Dispersed light also leads to thrombotic occlusion of the meningeal arteries in the parasagittal area. Among these arteries, thrombosis in pericallosal and penetrating arteries was present. Our experiment demonstrated that if a non-laser lamp is used, brain areas distant from the necrotic ring must be carefully investigated.

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.

Morphological changes in the brain during experimental hyponatraemia. Do vasopressin and gender matter?

Hyponatraemia is the most common electrolyte balance disorder occurring in hospitalized patients. The disease results frequently from inappropriate secretion of vasopressin (SIADH). It has been evidenced that the brain consequences of hyponatraemia are more dramatic in young females than in men or postmenopausal women. Since both vasopressin and oestrogen have been reported to inhibit ion fluxes essential for the adaptation of the brain to the lowering of serum sodium concentration, we sought to study the effect of acute and chronic hyponatraemia or hyponatraemia associated with vasopressin on brain morphology in male and female rats. Hyponatraemia was induced with vasopressin (AVP) or with desmopressin (dDAVP) in 12 male and 12 female adult Wistar rats for either 3 hours (acute) or 3.5 days (chronic). The brains of the animals with diagnosed hyponatraemia were fixed in 10% formalin and, following the standard procedure, stained with haematoxylin and eosin. Acute hyponatraemia resulted in white matter oedema with no obvious differences between genders or between groups with AVP- or dDAVP-induced hyponatraemia. Although in chronic hyponatraemia most neurons and astrocytic nuclei appeared to be normal, some neurons were swollen or ischaemic ("dark" neurons) and astrocytes showed a weak reaction. The most spectacular differences between males and females were found in the appearance of blood vessels. Swollen endothelial cells were observed more frequently in female than in male brains and in AVP- than in dDAVP-induced hyponatraemia. The widened Virchow-Robin spaces indicated perivascular oedema and blood-brain barrier damage. The results point to limited vascular adaptation to AVP-associated hyponatraemia in female gender.

Myelin composition of spinal cord in a model of amyotrophic lateral sclerosis (ALS) in SOD1G93A transgenic rats.

We present the results of biochemical and electron microscopic (EM) examinations of the spinal cord myelin from SOD1G93A transgenic Sprague Dawley rats in the early and late symptom-free period of the disease (60 and 93 days of life) and after four-leg paralysis has occurred (120 days of life). Biochemical and ultrastructural changes of myelin started already in the symptom-free period and become most pronounced in the paralyzed animals. Biochemical examinations indicated a decrease of lipids, phospholipids, cholesterol and cerebrosides. The pattern of particular phospholipids was in the normal range. A progressive decrease of the percentages of proteolipid, DM-20 and Wolfgram proteins was evident. Myelin basic proteins I and II were less affected. In EM,massive myelin disorganization was observed.

Morphological changes and selective loss of motoneurons in the lumbar part of the spinal cord in a rat model of familial amyotrophic lateral sclerosis (fALS).

Morphological alterations and the course of changes in motoneuron counts were studied by light microscopy (cresyl violet staining) in the L2/L3 region of the spinal cord of hemizygotic transgenic rats carrying the amyotrophic lateral sclerosis-associated mutant human gene for Cu,Zn superoxide dismutase (hSOD1G93A) and of their non-transgenic littermates. In 60-day old transgenic rats, a few ischaemic-looking alpha-motoneurons and occasional vacuolization and accumulation of tigroid in some of the cells were apparent. On day 93 of life more distinct cellular pathology was found in transgenic rats, including moderate gliosis, neuronophagy of alpha-motoneurons, and occasional neuronophagy of gamma-motoneurons. In 120-day-old transgenic rats, abundant gliosis and profound neuronophagy of alpha-motoneurons were observed combined with occasional neuronophagy of other cells. Some loss of alpha-motoneurons was also apparent in 120-day-old non-transgenic littermates of the transgenic rats. No difference in alpha-motoneuron and gamma-motoneuron counts was found between the rats on day 60 of life (early presymptomatic stage of the model disease in the transgenic rats). At 93 days of age (late presymptomatic stage), alpha-motoneuron count, but not gamma-motoneuron count, tended to be lower (p=0.06) in the transgenic rats. On day 120 of life (symptomatic stage), alpha-motoneuron count in the transgenic rats was about half that in their nontransgenic littermates (p<0.001); at this time point the relative decline in alpha-motoneuron number in the former was 57% (day 120 versus day 60; p<0.001). A smaller decline in alpha-motoneuron count was also found in nontransgenic rats (day 120 vs day 60: 24%, p<0.05); this was not associated with the emergence of neurological symptoms or distinct changes in the cell morphology of the spinal cord region studied.

Progression of morphological changes within CNS in a transgenic rat model of familial amyotrophic lateral sclerosis.

UNLABELLED: An analysis of the dynamics of histological and immunocytochemical changes in the CNS of a transgenic rat model of fALS in various periods of life was performed. Material was obtained from animals on the 60th day of age (4), 93rd day of age (3) and 120th presymptomatic day and from 3 animals in paretic stage of the disease. Formalin-fixed and paraffin-embedded slices were stained with HE and Klüver-Barrera method. Immunoreactions to GFAP, S-100, ferritin, neurofilament, ubiquitin, synaptophysin and tau protein were also performed. Within the brain tissues patchy neuronal loss and dark or ischaemic neurons were dispersed in cortical layers, CA1, CA3 and CA4 hippocampal areas and structures of the hemispheres and brain stem. In the spinal cord, numerous alpha motoneurons were dark or ischaemic. Vacuoles or small pale spots were visible in their cytoplasm. Microspongiosis surrounded some motoneurons, particularly cells subjected to neuronophagy. Neuronophagy, sporadically observed at the age of 60th day, was more extensive on the 93rd day of age, and at the age of 120 days already involved all interneurons of the anterior and posterior horns. In the immune reaction to neurofilament numerous fibres, often thick, fragmented or rosary-like, were observed. They were located within subcortical white matter, external and internal capsules, anterior horns of the spinal cord. Changes became more intensive with age. Astrocytic reactivity was weak in animals on the 60th and 93rd day of life. Non-numerous cells were immunoreactive to GFAP and S-100, although an increase of astrocytic nuclei was observed. On the 120th day of age and in symptomatic stage astrocytic hypertrophy and proliferation were intensive. But from the 60th day of age ubiquitin and tau protein immunopositive material was accumulated in the perinuclear area of astroglial cytoplasm. Immunoreaction of nerve cells to these proteins was negative. CONCLUSIONS: 1) In the subclinical stage of the disease the pathological process within the CNS takes place already on the 60th day of age and its intensity increases with age. 2) Morphological changes are not limited to motor neuronal cells. Various structures of the CNS are damaged. 3) Weak astroglial reaction probably depends on pathological accumulation of ubiquitin and tau protein in cytoplasm. 4) Astroglial cells are probably also a "target" for pathogenic factors in the rat model of fALS.

Ultrastructural changes in lumbar spinal cord in transgenic SOD1G93A rats.

The purpose of this study was to determine structural changes which trigger the onset and progression of amyotrophic lateral sclerosis in rats expressing a human SOD1 transgene with mutation G93A. Lumbar spinal cord of affected rats in early and late presymptomatic (PM, 60 and 93 days of age) and symptomatic (S, 120 days of age) stage of the disease were analyzed ultrastructurally. At 60 days the structure of lumbar spinal cord as well as alpha motoneurons type S and F appeared normal; however, careful examination revealed that approximately 15% of axons were filled with mitochondria that were abnormal in number, size and morphology. Grossly swollen mitochondria with disrupted cristae were a prominent feature in all large axons at 93 days of age. At this time swelling and dilated mitochondria were observed also in type S motoneurons, while type F had small, well preserved mitochondria. At symptomatic stage the alpha motoneurons showed moderate neuronal loss, mainly of the S type. The most interesting finding at this stage was the occurrence of motoneurons with morphological signs of apoptotic-like degeneration. Such apoptotic-like motoneurons were characterized by nuclear and cytoplasmic condensation, chromatin compaction and formation of uniformly dense, dark structures. Numerous axons with very dark, compact interior as well as apoptotic bodies were irregularly scattered throughout the neuropil. Our ultrastructural study indicates that dying motoneurons in transgenic mutant SOD1G93A rats exhibit reminiscent apoptotic morphology which is preceded by significant mitochondrial abnormalities mainly in proximal axons and S motoneurons. Different reaction of slow and fast motoneurons to degenerating factors requires further analysis.

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.

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.

Neuroprotective effects of the agonist of metabotropic glutamate receptors ABHxD-I in two animal models of cerebral ischaemia.

The neuroprotective efficacy of 2-aminobicyclo[2.1.1]hexane-2,5-dicarboxylic acid-I (ABHxD-I), a rigid agonist of metabotropic glutamate receptors, was studied using a 3-min global cerebral ischaemia model in Mongolian gerbils and the hypoxia/ischaemia model in 7-day-old rats. The effects on brain damage of ABHxD-I (30 mg/kg, intraperitoneally or 7.5 microg intracerebroventricularly) administered 30 min before global ischaemia or 30 min after hypoxia/ischaemia was evaluated 14 days after the insults. Treatment of adult gerbils with ABHxD-I injected i.c.v. but not systemically, prevented post-ischaemic hyperthermia and substantially reduced brain damage. These effects may reflect low permeability of the adult blood-brain barrier to ABHxD-I, and the role of reduced body and brain temperature in neuroprotection after its i.c.v. administration. ABHxD-I given either i.p. or i.c.v. to developing rats reduced brain damage by 55 and 37%, respectively, without affecting the body temperature. Due to immaturity and increased post-ischaemic permeability of the blood-brain barrier in developing rats, ABHxD-I may induce neuroprotection by direct interference with brain metabotropic glutamate receptors.

Behavioral evaluation of ischemic damage to CA1 hippocampal neurons: effects of preconditioning.

In Mongolian gerbils, global forebrain ischemia induces enhanced locomotor activity and the disruption of nest building immediately after the insult, followed by damage to hippocampal neurons developing 3 days later. Preconditioning by a brief episode of sublethal ischemia induces the protection of CA1 hippocampal neurons against a lethal ischemic insult. We examined how preconditioning with 2-min ischemia affects disturbances in the nest building behavior and locomotor activity induced by the injurious 3-min ischemia. Morphological examination confirmed that preconditioning significantly reduced neuronal damage in CA1 evoked by injurious ischemia. Behavioral studies demonstrated that preconditioning reduced the locomotor hyperactivity and latency in nest building after test ischemia, in comparison to sham or naive animals. The results indicate that the nest building test and measurement of locomotor activity may be used for an early in vivo prediction of the extent of ischemic brain damage and tolerance induced by ischemic preconditioning.

Inhibition of poly(ADP-ribose) polymerase activity protects hippocampal cells against morphological and ultrastructural alteration evoked by ischemia-reperfusion injury.

Poly(ADP-ribose) polymerase 1 (PARP-1 EC 2.4.2.30) is a nuclear enzyme that plays an important role in cell survival and death. PARP is involved in DNA repair machinery, however, massive DNA damage leads to over-activation of PARP-1 and to depletion of its substrate bNAD+ which causes cell death. Our previous study indicated that the PARP activity was significantly activated during ischemia-reperfusion injury. In this study we investigated the effect of PARP inhibitor, 3-aminobenzamide (3-AB) on intracellular organelles alteration. Gerbils were submitted to 3 and 10 min transient global ischemia followed by recirculation and survival for 1 till 7 days. The histological and electron microscopic examination indicated a pronounced protective effect of 3-AB on the swelling of astrocytes and neurons 1 day after 3 and 10 min ischemic insult. It decreased also the swelling of pericytes. 3-AB decreases evoked by ischemia swelling of mitochondria and Golgi apparatus. The significant ameliorating effect of 3-AB was also observed on the 7th day of reperfusion after 3 min ischemia and was also visible on the 1st day after 10 min ischemia. However, 7 days after prolonged 10 min ischemia almost all neurons in the CA1 hippocampal layer died and 3-AB was not able to protect these cells. In spite of that, 3-AB markedly decreased immunostaining of glial fibrillary acidic protein (GFAP), which was enhanced in the stratum: oriens, radiatum and lacunosum-moleculare at the 7th day after 10 min ischemia. These data indicated that inhibition of PARP may have a protective effect on neuronal cells affected by ischemia-reperfusion injury.

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.

Changes in the distribution of sugar receptors in blood vessels and cell elements of rat brain caused by photochemically induced focal ischaemia.

The lectin technique was applied to the histochemical studies of glycoconjugates comprising membranes of capillary endothelial cells and other cellular elements of the CNS. In the studies we used the following lectins: Concanavalin A (Con A), Bandeirea simplicifolia agglutinin (BS-I), Helix pomatia agglutinin (HPA), Ricinus communis agglutinin (RCA-120), Wheat germ agglutinin (WGA). We noticed changes in the localisation of the glycoconjugates, which are the result of cerebral ischaemia caused by focal irradiation of the left hemisphere of the brain after the previous intravenous injection of control animals with Rose bengal. The described changes seem to be the result of disturbances of the functioning of the blood-brain barrier, which was previously described (Gadamski et al. 2001). In this study, however, we have shown morphological and histochemical changes, which lead to the translocation of examined glycoconjugates within the appearing necrosis areas, around them and in the regions significantly distant from them. This refers to neurones, glial cells and endothelia of brain vessels. So we have described a clear diminution of Con A and HPA-positive receptors from neocortical and hippocampal neurones. The Con A receptors appeared however in morphologically changed, contracted capillaries and HPA receptors in macrophages and reactive cells of necrosis areas. In these areas the BSI-I receptors also appeared and additionally labelled astrocytes in the hippocampus and oligodendrocytes in the corpus callosum. The endothelial glycoconjugates visualised by RCA-120 and WGA also showed weakening of the histochemical reaction in the CNS vascular network and appeared in phagocytes and reactive astrocytes. The focal brain ischaemia induced this way, leading to disturbances in localisation and accumulation of the specific sugar receptors revealed by the lectin technique, determines the histochemical expression of the postischaemic changes in CNS.