α-synuclein antibody 5G4 identifies idiopathic REM-sleep behavior disorder in abdominal skin biopsies.

INTRODUCTION: Idiopathic REM-sleep behavior disorder (iRBD) is considered the most specific prodromal marker of Parkinson's disease (PD). With the need to improve early detection of prodromal α-synucleinopathies, several methods to identify peripheral α-synuclein (α-syn) pathology have been exploited in manifest and prodromal PD with varying diagnostic accuracy. Recently, a disease specific 5G4 antibody has been evaluated in skin biopsies of manifest PD patients. The aim of our study was to analyze the 5G4 α-syn immunoreactivity in skin biopsies of deeply phenotyped subjects with iRBD and controls. METHODS: The study cohort consisted of 28 patients with PD, 24 subjects with iRBD and 27 healthy controls, recruited from the CEGEMOD, PDBIOM and PARCAS cohorts. All subjects were deeply phenotyped and assessed for prodromal PD (pPD) probability based on MDS research criteria. Abdominal skin punch biopsies were processed and stained using a conformation specific 5G4 α-syn antibody as well as axonal markers SMI-31 and S100. RESULTS: 5G4-positivity was identified in 23/28 PD patients, 20/24 iRBD subjects and 8/27 healthy controls. Compared to healthy controls, sensitivity and specificity reached 83.33 % and 70.37 % for iRBD; and 82.14 % and 70.37 % for PD, respectively. 5G4-positivity rate in our study was irrespective of the calculated pPD probability of iRBD subjects. CONCLUSIONS: This work establishes the diagnostic yield of conformation specific 5G4 α-syn antibody testing in skin biopsies of subjects with pPD, specifically iRBD. The diagnostic accuracy for this method seems to be similar for both manifest and prodromal PD and is not dependent on the pPD probability ratios.

Influence of Escherichia coli infection on intestinal mucosal barrier integrity of germ-free piglets.

AIMS: We focused on investigating the influence of Escherichia coli (E. coli) on the intestinal barrier. MATERIAL AND METHODS: We studied changes in the distribution and secretory activities of goblet cells and enteroendocrine cells (EECs), as well as changes in the population of mast cells (MCs) in the jejunal and colonic mucosa of germ-free (GF) piglets as a healthy control group and GF piglets whose intestines were colonised with E. coli bacteria on day 5. KEY FINDINGS: The results suggest that the colon of GF piglets is more resistant and less prone to coliform bacterial infection compared to the jejunum. This can be confirmed by a lower degree of histopathological injury index as well as an improvement of the morphometric parameters of the colonic mucosa, together with a significantly increased (p < 0.05) expression of MUC1/EMA, and ZO-3. We also observed a significant decrease in the population of activated MCs (p < 0.001) and EECs (p < 0.001). These findings may indicate a rapid response and better preparation of the intestinal barrier for possible pathological attacks and the subsequent development of mucosal lesions during the development and progression of the intestinal diseases. SIGNIFICANCE: To date, gut-targeted therapeutic approaches that can modulate bacterial translocation and chronic inflammation are still in their infancy but represent one of the most promising areas of research for the development of new effective treatments or clinical strategies in the future. Therefore, a better understanding of these processes can significantly contribute to the development of these targeted strategies for disease prevention and treatment.

Programmed death ligand-1 expression and its association with the degree of differentiation and the presence of necrosis in non-small cell lung carcinoma.

The mechanisms underlying the expression of programmed death ligand-1 (PD-L1) in non-small cell lung carcinoma (NSCLC) are not yet fully clarified. In this study, surgical resections of 730 lung cancer patients with diagnosed NSCLC were analyzed. Results of PD-L1 immunohistochemistry (using clone 22C3) were correlated with clinicopathological variables including the degree of tumor differentiation and the presence of confluent areas of coagulative necrosis. PD-L1 immunohistochemistry was analyzed in tumor cells, whereas PD-L1 positivity was defined as membranous staining in ≥ 1 of tumor cells. A significantly higher proportion of PD-L1 positive cases was noted in poorly differentiated (grade 3) adenocarcinomas compared to better differentiated (grade 1 and grade 2) subtypes (63.8 % vs. 28.7 %; p < 0.001). Contrary to this, better differentiated (keratinizing) and less differentiated (non-keratinizing) squamous cell carcinoma subtypes were found to have a similar proportion of PD-L1 positive cases (51.4 % vs. 55.8 %; p = 0.570). High levels of PD-L1 expression significantly correlated with the presence of necrosis in NSCLC: seventy-nine of 109 NSCLC cases with the presence of necrosis were PD-L1 positive compared to 256 out of 621 NSCLC without necrosis (72.5 % vs. 41.2 %; p < 0.001). High PD-L1 expression was not positively correlated with age, gender, and advanced T stage but a significant association between PD-L1 positivity and higher N stage was observed (p < 0.001) in NSCLC patients. In conclusion, the proportion of PD-L1 positive cases is higher only in poorly differentiated NSCLC of the adenocarcinoma type. A significantly higher overall rate of PD-L1 positive cases was noted in NSCLC with the presence of necrosis. Further investigation is suggested to elucidate the intricated interconnections between the plethora of hypoxic biomarkers and immunological factors in different types and subtypes of NSCLC.

Remote conditioning as protective strategy influencing ubiquitin-mediated stress response in the rabbit spinal cord after ischemia/reperfusion.

The aim of presented study was to investigate the model of non-invasive method of remote conditioning induced by compression of left forelimb with a tourniquet in three cycles of 2 min of ischemia each followed by 2 min of reperfusion and its influence on the rabbit spinal cord ischemia/reperfusion injury via ubiquitin-mediated stress response. Ubiquitin immunoreaction in spinal cord motor neurons as well as detection of neuronal survival in ventral horns of spinal cord were evaluated. Significantly increased (p < 0.001) number of ubiquitin positive neurons was registered in all remote conditioned groups versus both spinal cord ischemia (SC-ischemia) groups. Our results indicate that remote conditioning significantly attenuated degeneration of motor neurons in all conditioned groups versus SC-ischemia groups in each time point. According to our results, we concluded that the remote conditioning induced by transient limb ischemia is relevant stimulus that provides potent neuroprotection in a model of spinal cord ischemia/ reperfusion injury.

Immunohistochemical visualisation of the enteric nervous system architecture in the germ-free piglets.

The enteric nervous system (ENS), considered as separate branch of the autonomic nervous system, is located throughout the length of the gastrointestinal tract as a series of interconnected ganglionic plexuses. Recently, the ENS is getting more in the focus of gastrointestinal research. For years, the main interest and research was aimed to the enteric neurons and their functional properties in normal conditions, less attention has been paid to the germ-free animals. Germ-free (GF) piglets have clear microbiological background and are reared in sterile environment. GF piglets are regarded as clinically relevant models for studying of human diseases, as these piglets' manifest similar clinical symptoms to humans. In this study we briefly summarised the main characteristics in immunohistochemical distribution of ENS elements in the wall of jejunum and colon of germ-free piglets.

Induction of ischemic tolerance by remote perconditioning or postconditioning as neuroprotective strategy for spinal cord motor neurons.

AIMS: The study is focused on the investigation of the mechanisms leading to ischemic tolerance acquisition in the spinal cord neurons via application of non-invasive method of remote conditioning. MATERIAL AND METHODS: We have verified the possibility of neuroprotection of spinal cord in rabbit by using remote perconditioning (PerC) applied during last 12 min of spinal cord ischemia (SC-ischemia) or postconditioning (PostC) applied after 1st (early) or 3rd (late) h of reperfusion. Spinal cord ischemia was induced by occlusion of the aorta below the left renal artery for 20 min. Reperfusion period was 24 or 72 h. Remote conditioning was induced by compression of left forelimb with a tourniquet in 3 cycles of 2 min of ischemia, each followed by 2 min of reperfusion. Damaged neurons were detected by Fluoro Jade B method and the modified Tarlov score was used for functional assessment. KEY FINDINGS: The remote conditioning significantly attenuated degeneration of motor neurons in all remote conditioned groups versus both SC-ischemia groups. We detected significant changes in number of Hsp70 positive motor neurons. At 72time point, in the group with remote late PostC we observed significant increase (p < 0.001) of Hsp70 positive motor neurons versus SC- ischemia group and sham control. There was a trend towards improvement of hindlimbs movement. SIGNIFICANCE: This study showed the effectiveness of remote conditioning as a neuroprotective strategy, evidenced by induction of ischemic tolerance leading to decrease of motor neuron degeneration.

Bradykinin and noradrenaline preconditioning influences level of antioxidant enzymes SOD, CuZn-SOD, Mn-SOD and catalase in the white matter of spinal cord in rabbits after ischemia/reperfusion.

The aim of present work is to assess the effects of bradykinin (Br) or noradrenaline (Nor) preconditioning to the levels of antioxidant enzymes: superoxide dismutase (SOD), copper, zinc superoxide dismutase (CuZn-SOD), manganese superoxide dismutase (Mn-SOD) and catalase in ischemia/reperfusion (I/R) model in the rabbit spinal cord white matter as well as effect on glial fibrillary acidic protein (GFAP) and ubiquitin immunoreaction in glial cells. Rabbits were preconditioned by intraperitoneal single dose of Br or Nor 48 h prior to 20 min of ischemia followed by 24 or 48 h of reperfusion. White matter of L3-L6 spinal cord segments was used for comparison of antioxidant enzyme levels in sham control, ischemic groups and four preconditioned groups. The total SOD level in the Br or Nor preconditioned groups after 48 h of reperfusion was increased vs Br or Nor preconditioned groups after 24 h of reperfusion. The comparison among the ischemic group vs Br preconditioned (P<0.05), and Nor preconditioned (P<0.001) groups after 48 h of reperfusion, showed statistically significant decrease of Mn-SOD activity. Tissue catalase level activity was significantly decreased in the Br preconditioned group after 48 h of reperfusion (P<0.05) and Nor preconditioned groups after 24 h of reperfusion (P<0.001) and also after 48 h of reperfusion (P<0.001), in comparison to ischemic group after 48 h of reperfusion. Significantly decreased tissue catalase activity (P<0.05) in both Nor preconditioned groups after 24 or 48 h of reperfusion was measured vs Br preconditioned group after 48 h of reperfusion. According to our results, in the white matter, activation of stress proteins in glial cells, as well as antioxidant enzymes levels, were influenced by pharmacological preconditioning followed by 20 min of ischemia and 24 or 48 h of reperfusion. These changes contribute to ischemic tolerance acquisition and tissue protection from oxidative stress during reperfusion period.

The effect of Betanin parenteral pretreatment on Jejunal and pulmonary tissue histological architecture and inflammatory response after Jejunal ischemia-reperfusion injury.

Intestinal ischemic-reperfusion (IR) injury has detrimental effects on both local and distant organs in the body. Betanin is known for its antioxidant properties, and it is found mostly in vegetables. Therefore, the aim of the present study was to test the hypothesis that betanin administration prior intestinal IR, may be beneficial in protecting jejunal mucosa and lung parenchyma against IR damage. Male specific pathogen-free Charles River Wistar rats were used (n = 42). Betanin (50 mg/kg) was administered intraperitoneally 30 min before ischemia of the superior mesenteric artery lasting 1 h, followed by 1, 4 and 24 h of reperfusion. Immunohistochemical as well as histomorphometrical analysis indicated a protective effect of betanin pretreatment on jejunal tissue. Regarding morphometrical analysis betanin significantly (p < 0.01) augments intestinal villus height after 24 of reperfusion comparing to early stages. Betanin application reduced number of mast cells population in early reperfusion periods (p < 0.05). The protective effect of betanin on lung parenchyma, was detected in late reperfusion period (24 h) with improvement of histopathological injury index and morphometric analysis (p < 0.001 for both). The improvement of histopathological injury index (p < 0.001) and morphometric analysis (p < 0.001) during the late reperfusion period, suggests a protective effect of betanin on lung parenchyma. Moreover, suppression of the inflammatory response was mirrored by the reduction of myeloperoxidase (MPO) positive cells within lung parenchyma after 1 and 4 h of reperfusion (p < 0.001). Especially, during the first 4 h of reperfusion after betanin administration, a reduction of 74% of the polymorphonuclear neutrophils infiltration (MPO positive cell population) and of a nearly 46% of active MCs was observed. Upon morphometric examination, the lung histological architecture after 24 h of reperfusion appeared to be almost 100% better following betanin treatment, with 25% thinner interalveolar septa and 20% larger alveolar surface for respiratory gas exchange. The results suggest that betanin pretreatment protects the jejunal mucosa and the lung parenchyma, as well as reduces the inflammatory cell density after intestinal IR injury.

Ubiquitin and endogenous antioxidant enzymes participate in neuroprotection of the rabbit spinal cord after ischemia and bradykinin postconditioning.

The aim of this study was to investigate neuroprotective effect of bradykinin postconditioning on the rabbit spinal cord after 20 min of ischemia and 3 days of reperfusion. Bradykinin was administered by single i.p. application at 1, 6, 12 or 24 h after ischemia. Assessment of neurological function of hind limbs (Tarlov score) was estimated. Quantitative analysis was evaluated by Fluoro Jade B method, NeuN and ubiquitin immunohistochemistry in anterior horn neurons of the spinal cord. Histomorphologically distribution of ubiquitin and endogenous antioxidant enzymes (SOD1, SOD2, catalase) immunoreaction was described. Bradykinin postconditioning showed decreased number of degenerated neurons, increased number of surviving neurons and increase in number of ubiquitin positive neurons in all bradykinin postconditioned groups versus ischemia/reperfusion group. According to our results bradykinin postconditioning applied 24 h after ischemia significantly decreased (p < 0.001) number of degenerated neurons versus ischemia/reperfusion group. The least effective time window for bradykinin postconditioning was at 12 h after ischemia. Tarlov score was significantly improved (p < 0.05) in groups with bradykinin postconditioning applied 1, 6 or 24 h after ischemia versus ischemia/reperfusion group. Tarlov score in group with bradykinin application 12 h after ischemia was significantly decreased (p < 0.05) versus sham control group. Neuronal immunoreaction of ubiquitin, SOD1, SOD2 and catalase influenced by bradykinin postconditioning was dependent on neuronal survival or degeneration. In conclusion, bradykinin postconditioning showed protective effect on neurons in anterior horns of the rabbit spinal cord and improved motor function of hind limbs.

Quercetin protects jejunal mucosa from experimental intestinal ischemia reperfusion injury by activation of CD68 positive cells.

The aim of our study was to analyse the possible protective effect of quercetin application during the jejunal ischemia-reperfusion injury (IRI) in rats. Quercetin was administered intraperitoneally 30min before 1h ischemia of superior mesenteric artery with following 24h lasting reperfusion period. The male specific pathogen-free (SPF) Charles River Wistar rats were used. In the group with applied quercetin, the significantly increased (p<0.001) levels of anti-inflammatory cytokine IL10 were observed both in the blood serum and jejunal tissue. The improvement of the mucosal tissue morphology and proliferating and DNA repairing cell number measured by PCNA activity were recorded by more than 30% higher in the quercetin group. Simultaneously, significant elongation of the intestinal glands (p<0.001) and increase in the number of CD68-positive cells in the lamina propria mucosae (p<0.001) in comparison with control group were found. Based on our results, the preventive application of quercetin before induction of jejunal IRI stimulates faster jejunal mucosa restoration and it seems to have immunomodulatory and anti-inflammatory effects as well. CD68-positive macrophages could have crucial role in this process since they work as both growth factor and cytokine producers.

Prevalence of Prodromal Parkinson's Disease as Defined by MDS Research Criteria among Elderly Patients Undergoing Colonoscopy.

BACKGROUND: Gastrointestinal symptoms are a well-recognized and common premotor feature of Parkinson's disease (PD). Moreover, multiple studies have assessed the value of colonic α-synuclein as a potential marker of prodromal PD. Recently, the International Parkinson and Movement Disorders Society (MDS) defined research criteria for prodromal PD. OBJECTIVE: The aim of our study was to test the MDS research criteria in patients undergoing diagnostic colonoscopies as potential candidates for inclusion in prospective trials evaluating colonic biopsies as a potential biomarker of prodromal PD. METHODS: We evaluated elderly patients without manifest parkinsonism undergoing diagnostic colonoscopies. During the study we assessed all risks and prodromal markers of the MDS research criteria, excluding radiotracer imaging and genetic testing. RESULTS: The mean age of the 100 enrolled patients was 61.6±9.7 years; 42 were men. The most common prodromal marker in our cohort was constipation (40%), followed by MDS-UPDRS part III scores of >6 points, excluding action tremor items (39%) and hyposmia (37%). Substantia nigra hyperechogenicity was identified in 9%, and polysomnography confirmed REM sleep behavior disorder in 2% of the patients. Five of the 100 enrolled patients (5%) fulfilled the criteria for probable prodromal PD, while another 3 patients met the 50% probability threshold. CONCLUSIONS: Our findings suggest, that the prevalence of prodromal PD in patients undergoing diagnostic colonoscopies may be higher compared to the general elderly population, although this should be confirmed in further studies including also matched controls not undergoing colonoscopy. The real prevalence of prodromal PD in this cohort will have to be confirmed in longitudinal follow-up. Patients undergoing diagnostic colonoscopies may be good candidates for multistep screening and inclusion in prospective trials.

Bradykinin preconditioning affects the number of degenerated neurons and the level of antioxidant enzymes in spinal cord ischemia in rabbits.

Bradykinin preconditioning has been used for acquisition of tolerance after spinal cord ischemia. Rabbits were preconditioned intraperitoneally with bradykinin 48 h prior to 20 min of abdominal aorta ligation followed by 24 and 48 h of reperfusion. The activities of SOD and catalase were measured and Fluoro Jade B (FJB)-positive degenerated neurons were evaluated. The outcomes of Tarlov scoring system used to assess neurological functions showed significant improvement in bradykinin groups compared to the ischemic group. The number of FJB-positive degenerated neurons was decreased in ventral horns of both bradykinin groups. Significantly decreased activities of total SOD and mitochondrial Mn-SOD were also detected in both bradykinin groups versus ischemic group while CuZn-SOD and catalase activities were significantly decreased only in the bradykinin group after 24h of reperfusion versus ischemic group. These findings suggest that one of the possibilities of the neuroprotective effect of delayed bradykinin preconditioning against spinal cord ischemic injury could be realized by mitochondrial protection and decreased synthesis of Mn-SOD as well as by promotion of neuronal survival.

Response of ependymal progenitors to spinal cord injury or enhanced physical activity in adult rat.

Ependymal cells (EC) in the spinal cord central canal (CC) are believed to be responsible for the postnatal neurogenesis following pathological or stimulatory conditions. In this study, we have analyzed the proliferation of the CC ependymal progenitors in adult rats processed to compression SCI or enhanced physical activity. To label dividing cells, a single daily injection of Bromo-deoxyuridine (BrdU) was administered over a 14-day-survival period. Systematic quantification of BrdU-positive ependymal progenitors was performed by using stereological principles of systematic, random sampling, and optical Dissector software. The number of proliferating BrdU-labeled EC increased gradually with the time of survival after both paradigms, spinal cord injury, or increased physical activity. In the spinal cord injury group, we have found 4.9-fold (4 days), 7.1-fold (7 days), 4.9-fold (10 days), and 5.6-fold (14 days) increase of proliferating EC in the rostro-caudal regions, 4 mm away from the epicenter. In the second group subjected to enhanced physical activity by running wheel, we have observed 2.1-2.6 fold increase of dividing EC in the thoracic spinal cord segments at 4 and 7 days, but no significant progression at 10-14 days. Nestin was rapidly induced in the ependymal cells of the CC by 2-4 days and expression decreased by 7-14 days post-injury. Double immunohistochemistry showed that dividing cells adjacent to CC expressed astrocytic (GFAP, S100beta) or nestin markers at 14 days. These data demonstrate that SCI or enhanced physical activity in adult rats induces an endogenous ependymal cell response leading to increased proliferation and differentiation primarily into macroglia or cells with nestin phenotype.

Bradykinin postconditioning protects pyramidal CA1 neurons against delayed neuronal death in rat hippocampus.

AIMS: The present study was undertaken to evaluate possible neuroprotective effect of bradykinin against delayed neuronal death in hippocampal CA1 neurons if applied two days after transient forebrain ischemia in the rat. METHODS: Transient forebrain ischemia was induced in male Wistar rats by four-vessel occlusion for 8 min. To assess efficacy of bradykinin as a new stressor for delayed postconditioning we used two experimental groups of animals: ischemia 8 min and 3 days of survival, and ischemia 8 min and 3 days of survival with i.p. injection of bradykinin (150 microg/kg) applied 48 h after ischemia. RESULTS: We found extensive neuronal degeneration in the CA1 region at day 3 after ischemia/reperfusion. The postischemic neurodegeneration was preceded by increased activity of mitochondrial enzyme MnSOD in cytoplasm, indicating release of MnSOD from mitochondria in the process of delayed neuronal death. Increased cytosolic cytochrome c and subsequently caspase-3 activation are additional signs of neuronal death via the mitochondrial pathway. Bradykinin administration significantly attenuated ischemia-induced neuronal death, and also suppressed the release of MnSOD, and cytochrome c, and prevented caspase-3 activation. CONCLUSIONS: Bradykinin can be used as an effective stressor able to prevent mitochondrial failure leading to apoptosis-like delayed neuronal death in postischemic rat hippocampus.

Effect of antioxidant treatment in global ischemia and ischemic postconditioning in the rat hippocampus.

Ischemic postconditioning is a very effective way how to prevent delayed neuronal death. Effect of Ginkgo biloba extract (EGb 761; 40 mg/kg) posttreatment was studied on the rat model of transient forebrain ischemia and ischemia/postconditioning. Global ischemia was produced by four-vessel occlusion in Wistar male rats. Two experimental protocols were used: (a) 10 min of ischemia/7 days of reperfusion with or without EGb 761 treatment or (b) 10 min of ischemia/2 days of reperfusion/5 min of ischemia (postconditioning), following 5 days of reperfusion. EGb 761 was applied as follows: 30 min before 10 min of ischemia then 5 h, 1 and 2 days after 10 min of ischemia. Fluoro Jade B, marker for neuronal degeneration, was used for quantitative analysis of the most vulnerable hippocampal CA1 neurons. Cognitive and memory functions were tested by Morris water maze, as well. Administration of EGb 761 30 min before 10 min of ischemia or 5 h after ischemia has rather no protective effect on neuronal survival in CA1 region. Ten minutes of ischemia following ischemic postconditioning after 2 days of reperfusion trigger a significant neuroprotection of CA1 neurons, but it is abolished by EGb 761 posttreatment. Ischemia/postconditioning group showed a significant improvement of learning and memory on the seventh day of reperfusion. Protection of the most vulnerable CA1 neurons after ischemia/postconditioning is abolished by exogenous antioxidant treatment used in different time intervals after initial ischemia. Moreover, combination of EGb 761 administration with repeated stress (5 min ischemia used as postconditioning) causes cumulative injury of CA1 neurons.

Postconditioning and anticonditioning: possibilities to interfere to evoked apoptosis.

The aim of this study was to validate the ability of postconditioning, used 2 days after kainate intoxication, to protect selectively vulnerable hippocampal CA1 neurons against delayed neuronal death. Kainic acid (8 mg/kg, i.p.) was used to induce neurodegeneration of pyramidal CA1 neurons in rat hippocampus. Fluoro Jade B, the specific marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 days after intoxication without and with delayed postconditioning (norepinephrine, 3.1 mumol/kg i.p., 2 days after kainate administration) and anticonditioning (Extract of Ginkgo biloba, 40 mg/kg p.o used simultaneously with kainate). Morris water maze was used on 6th and 7th day after kainate to test learning and memory capabilities of animals. Our results confirm that postconditioning if used at right time and with optimal intensity is able to prevent delayed neuronal death initiated not only by ischemia but kainate intoxication, too. The protective effect of repeated stress-postconditioning was suppressed if extract of Ginkgo biloba (EGb 761, 40 mg/kg p.o.) has been administered together with kainic acid. It seems that combination of lethal stress and antioxidant treatment blocks the activation of endogenous protecting mechanism known as ischemic tolerance, aggravates neurodegeneration and, after repeated stress is able to cause cumulative damage. This observation could be very valuable in situation when the aim of treatment is elimination of unwanted cell population from the organism.

Effect of noradrenalin and EGb 761 pretreatment on the ischemia-reperfusion injured spinal cord neurons in rabbits.

Short term sublethal ischemia or ischemic preconditioning gives protection to the neurons against subsequent lethal ischemic attack. This so-called ischemic tolerance can also be provided by certain drugs. We examined the effect of noradrenalin and EGb 761 on the spinal cord neurons injured by 30 min occlusion of abdominal aorta in rabbits. The animals survived 48 and 72 h. Degenerated neurons were visualized by Fluoro Jade B method, viable neurons were demonstrated immunohistochemically with NeuN and ubiquitin antibodies. The rabbits with noradrenalin administration 48 h before 30 min of ischemia and 48/72 h of reperfusion, showed significant increase of degenerated Fluoro Jade B labeled neurons. Animals of both groups were paraplegic. Rabbits pretreated 7 days with EGb 761 prior to 30 min of ischemia and with 48/72 h of reperfusion revealed significant decrease of Fluoro Jade B-positive neurons when compared with the groups with 30 min of ischemia followed by 48/72 h of reperfusion. In the NeuN sections, the number of viable neurons was moderately decreased. These animals showed no paraplegia. Ubiquitin aggregates occurred in the cytoplasm of degenerated neurons in the sections of rabbits preconditioned with noradrenalin 48 h prior to 30 min of ischemia and followed by 48 h of reperfusion while after 72 h of reperfusion, shrunk light shadows without ubiquitin reaction were visible. Our results indicate that EGb 761 could be involved in protection of spinal cord neurons against ischemic injury while effect of noradrenalin is not unambiguous.

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment.

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min. 2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region. 3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group. 4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare. 5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.

Extract EGb 761 pretreatment limits ubiquitin positive aggregates in rabbit spinal cord neurons after ischemia-reperfusion.

1. Ubiquitin immunohistochemistry was used for investigation of time dependent changes of ubiquitin in the nerve cells reacting to ischemic/reperfusion damage. In the rabbit spinal cord ischemia model a period of 30 min ischemia followed by 24 and 72 h of reperfusion caused neuronal degeneration selectively in the ventral horn motor neurons as well as interneurons of the intermediate zone. 2. Ubiquitin aggregates were accumulated in the neurons of lamina IX and the neurons of intermediate zone destined to die 72 h after 30 min of the spinal cord ischemia. 3. The activation of ubiquitin hydrolytic system is related to a defective homeostasis and could trigger different degenerative processes. Having in mind this, we used EGb 761 to rescue the motor neurons and interneurons against ischemia/reperfusion damage. Our results show that after 30 min of ischemia and 24 or 72 h of reperfusion with EGb 761 pre-treatment for 7 days the vulnerable neurons in the intermediate zone and lamina IX exhibit marked elevation of ubiquitin-positive granules in the cytoplasm, dendrites and nuclei. Abnormal protein aggregates have not been observed in these cells. 4. The rabbits were completely paraplegic after 30 min of ischemia and 24 or 72 h of reperfusion. However, after 7 days EGb 761 pre-treatment, 30 min of ischemia and 24 or 72 h of reperfusion the animals did not show paraplegia. 5. Evaluated ubiquitin-positive neurons of the L(5)-L(6) segments showed significant decrease in number and significant increase of density after 30 min of ischemia followed by 24 h and mainly 72 h of reperfusion. Ubiquitin immunohistochemistry confirmed the protective effect of EGb 761 against ischemia/reperfusion damage in the rabbit spinal cord.