George Karkavelas

Erythropoietin prevents long-term sensorimotor deficits and brain injury following neonatal hypoxia-ischemia in rats

Perinatal asphyxia accounts for behavioral dysfunctions that often manifest as sensorimotor, learning or memory disabilities throughout development and into maturity. Erythropoietin (Epo) has been shown to exert neuroprotective effects in different models of brain injury including experimental models of perinatal asphyxia. However, the effect of Epo on functional abilities following cerebral hypoxia-ischemia (HI) in neonatal rats is not known. The aim of the present study is to investigate the effect of Epo on sensorimotor deficits and brain injury induced by hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia. Epo was administered as a single dose immediately after the hypoxic insult (2000 U/kg). The neuroprotective effect of Epo was evaluated at postnatal day 42 by using a battery of behavioral tests and histological analysis. The results of the present study suggest that Epo treatment immediately after HI insult significantly facilitated recovery of sensorimotor function. Consistently, histopathological evaluation demonstrated that Epo significantly attenuated brain injury and preserved the integrity of cerebral cortex. These findings indicate that long-term neuroprotective effect of Epo on neonatal HI-induced brain injury might be associated with the preservation of sensorimotor functions.

Phase II Study of Neoadjuvant Imatinib in Glioblastoma: Evaluation of Clinical and Molecular Effects of the Treatment

Purpose: Phase I-II studies indicate that imatinib is active in glioblastoma multiforme. To better understand the molecular and clinical effects of imatinib in glioblastoma multiforme, we conducted a neoadjuvant study of imatinib with pretreatment and posttreatment biopsies. Experimental Design: Patients underwent a computerized tomography-guided biopsy of their brain tumors. If diagnosed with glioblastoma multiforme, they were immediately treated with 7 days of imatinib 400 mg orally twice daily followed by either definitive surgery or re-biopsy. Pretreatment and posttreatment tissue specimens were tested by immunohistochemistry for Ki67 and microvessel destiny, and posttreatment specimens were analyzed for the presence of intact imatinib in tissue. Furthermore, pretreatment and posttreatment pairs were analyzed by Western blotting for activation of platelet-derived growth factor receptor, epidermal growth factor receptor (EGFR), phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase signaling pathways. Pharmacokinetic studies were also done. Results: Twenty patients were enrolled. Median survival was 6.2 months. Intact imatinib was detected in the posttreatment tissue specimens using mass spectrometry. There was no evidence of a drug effect on proliferation, as evidenced by a change in Ki67 expression. Biochemical evidence of response, as shown by decreased activation of AKT and mitogen-activated protein kinase or increased p27 level, was detected in 4 of 11 patients with evaluable, matched pre- and post-imatinib biopsies. Two patients showed high-level EGFR activation and homozygous EGFR mutations, whereas one patient had high-level platelet-derived growth factor receptor-B activation. Conclusions: Intact imatinib was detected in glioblastoma multiforme tissue. However, the histologic and immunoblotting evaluations suggest that glioblastoma multiforme proliferation and survival mechanisms are not substantially reduced by imatinib therapy in most patients. (Clin Cancer Res 2009;15(19):6258–66)

Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

Background Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations. Methodology/Principal Findings Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect ≤1% mutant cells, provided that samples yielded cycle thresholds (Ct) <29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%). Conclusions/Significance Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.

Hypoxia-ischemia affects erythropoietin and erythropoietin receptor expression pattern in the neonatal rat brain

Erythropoietin (EPO), known for its role in erythroid differentiation, has been suggested to have non-hematopoietic functions in the brain, especially during development. In the present study, we investigated the expression of erythropoietin and erythropoietin receptor (EPOR) in the developing rat brain following hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia, and their brains were examined immediately, 24 h or 4 days after hypoxia-ischemia. RT-PCR and Western blot analysis revealed that hypoxia-ischemia only marginally affected EPO expression. Immunohistochemical study of brains 4 days after hypoxia showed that 60 min of hypoxia (resulting in cortical infarction and severe neuronal loss in other regions) led to the increased EPO immunoreactivity, especially in the boundaries of the damaged cerebral cortex, associated with astrocytosis. In contrast, EPOR was dramatically upregulated within 24 h after hypoxia-ischemia. These results suggest that there is a rapid response of EPOR to the hypoxic-ischemic stimulus, which seems to precede that of EPO, leading to the hypothesis that the EPO/EPOR system is implicated in the processes of neuroprotection from hypoxia-ischemia.

Effect of ketamine on hypoxic-ischemic brain damage in newborn rats

The present study tests the hypothesis that ketamine, a dissociative anesthetic known to be a non-competitive antagonist of the NMDA receptor, will attenuate hypoxic-ischemic damage in neonatal rat brain. Studies were performed in 7-day-old rat pups which were divided into four groups. Animals of the first group, neither ligated nor exposed to hypoxia, served as controls. The second group was exposed to hypoxic-ischemic conditions and sacrificed immediately afterwards. Animals of the third and fourth groups were treated either with saline or ketamine (20 mg/kg, i.p.) in four doses following hypoxia. Hypoxic-ischemic injury to the left cerebral hemisphere was induced by ligation of the left common carotid artery followed by 1 h of hypoxia with 8% oxygen. Measurements of high energy phosphates (ATP and phosphocreatine) and amino acids (glutamate and glutamine) and neuropathological evaluation of the hippocampal formation were used to assess the effects of hypoxia-ischemia. The combination of common carotid artery ligation and exposure to an hypoxic environment caused major alterations in the ipsilateral hemisphere. In contrast, minor alterations in amino acid concentrations were observed after the end of hypoxia in the contralateral hemisphere. These alterations were restored during the early recovery period. Post-treatment with ketamine was associated with partial restoration of energy stores and amino acid content of the left cerebral hemisphere. Limited attenuation of the damage to the hippocampal formation as demonstrated by a reduction in the number of damaged neurons was also observed. These findings demonstrate that systemically administered ketamine after hypoxia offers partial protection to the newborn rat brain against hypoxic-ischemic injury.

Neuroprotective effect of long-term MgSO4 administration after cerebral hypoxia-ischemia in newborn rats is related to the severity of brain damage.

Previous studies have shown contradictory results regarding magnesium-mediated neuroprotection in animal models of perinatal asphyxia. The aim of this study is to investigate the e fects of MgSO4 postasphyxial treatment on hypoxia-ischemia (HI)—induced brain injury in neonatal rats and the possibility that this e fect is related to the severity of brain damage. Seven-day-old rats underwent unilateral carotid artery ligation followed by 1 or 2 hours of hypoxia (8% O2) and MgSO4 administration. Adenosine triphosphate/phosphocreatine and glutamate/glutamine measurements and neuropathological evaluation of the hippocampus were used to assess the e fects of HI and MgSO4. HI caused time-dependent changes in energy stores, amino acid concentrations, and brain damage. Administration of MgSO4 after 1 hour but not after 2 hours of hypoxia resulted in significant prevention of HI-induced brain injury. MgSO4 administration results in a significant protection against moderate HI-induced brain damage, whereas it fails to offer a similar effect against severe brain damage.

Evaluation of Long-Lasting Sensorimotor Consequences following Neonatal Hypoxic-Ischemic Brain Injury in Rats: The Neuroprotective Role of MgSO4

Perinatal asphyxia (PA) is a major determinant for long-term sensorimotor and locomotor deficits. The model of neonatal hypoxia-ischemia (HI) in 7-day-old rats produces sensorimotor cortex, thalamus and striatum injury, which are all critical for the maintenance of sensory motor function. The aim of this study was to evaluate the long-term neurodevelopmental disturbances in the above experimental model and to assess the neuroprotective effect of MgSO4 in terms of long-term behavioral and morphological changes. Seven-day-old rats were separated into three groups: A (control), neither ligated nor exposed to hypoxia; B (HI/MgSO4) ligated, exposed to hypoxia and treated with MgSO4 (2 g/kg b.w., i.p.), and C (HI) ligated and exposed to hypoxia. At the age of 42 days, the behavior of the rats was evaluated using 5 sensorimotor tests. Muscle power, motor coordination, reflexes, and limb placing were tested to different sensory stimuli. The study was completed with the histopathological evaluation of brain tissue damage. In all individual tests the HI-treated rats performed significantly worse than the control and MgSO4-treated rats and this difference was more pronounced in the limb placing tests. Additionally, neonatal HI resulted in extensive neuronal damage that was limited after MgSO4 administration. Behavioral alterations represent a useful endpoint for studying the consequences of a perinatal HI insult and the efficacy of potential neuroprotective treatments. MgSO4 administration resulted in prevention of HI-induced sensorimotor deficits and brain injury.

Erythropoietin prevents hypoxia/ischemia-induced DNA fragmentation in an experimental model of perinatal asphyxia

Erythropoietin (EPO) prevents neuronal damage following ischemic, metabolic and excitotoxic stress. Recent studies have shown that EPO plays a significant role in the developing brain. The present study investigates the effect of EPO administration on hypoxic-ischemic brain injury and the possibility that its neuroprotective action may be associated with anti-apoptotic activity. Seven-day-old rats were treated with EPO (2000 U/kg) and subjected to a modified Levine procedure. EPO administration before the hypoxic-ischemic insult significantly reduces the severity of brain damage and improved the short-term functional brain recovery. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and DNA electrophoresis displayed no evidence of DNA fragmentation in EPO-treated animals. These results suggest that EPO might protect the neonatal rat brain by anti-apoptotic mechanisms.

Radiation and Concomitant Weekly Administration of Paclitaxel in Patients with Glioblastoma Multiforme. A Phase II Study

The present study was conducted to evaluate the activity and toxicity profile of radiation (RT) and concomitant chemotherapy in patients with glioblastoma multiforme (GBM). Thirty-nine patients were treated postoperatively with RT and concomitant administration of paclitaxel. Cranial irradiation was initiated 2–3 weeks postoperatively and was administered in 2.0 fractions, one fraction per day, for 5 consecutive days per week, to a total of 60 Gy. Paclitaxel was delivered at a dose of 100 mg/m2 over 3-h once weekly for 6 weeks.Thirty-three patients received all 6 cycles of paclitaxel according to the protocol. Totally, 217 cycles were delivered all of them at full dose. The median relative dose intensity of paclitaxel was 1 (range 0.88–1.1). Three (7.5%) patients achieved complete and 9 (23%) partial response, while 12 (30.5%) patients demonstrated stabilization of the disease. Side effects from combined chemoradiotherapy were mainly mild. Grade III toxicity included infection (7.5%) and alopecia (5%). Median time to progression was 6 (range 0.9–27) months and median survival 10.7 (range 0.9–39.5+) months.The present study has clearly shown that 100 mg/m2 of paclitaxel in 1-h infusion weekly can be safely given concomitantly with RT in patients with GBM with manageable toxicity. However, the efficacy of this combined modality treatment does not appear to be superior to that of RT alone.

Manifestations of pilocytic astrocytoma: a pictorial review

BackgroundPilocytic astrocytoma can be challenging to diagnose.MethodsIts clinical presentations can differ, directly related to its size and location, and are relatively unreliable. Similarly, imaging findings also vary with the location of the pilocytic astrocytoma.ResultsThis review provides an overview of the different imaging findings regarding pilocytic astrocytomas using both conventional and advanced magnetic resonance imaging sequences according to tumour location; the findings are strongly related to the tumour’s tendency to infiltrate surrounding structures, being able to carry out gross total resection, and finally the prognosis.ConclusionsKnowledge of these imaging manifestations of pilocytic astrocytoma may be helpful to arrive at an accurate diagnosis.Teaching PointsTo recognise the various imaging findings of pilocytic astrocytoma on both conventional and advanced magnetic resonance imaging sequences.To identify the characteristic imaging findings according to tumour location.To discuss the relevant differential diagnoses of pilocytic astrocytoma in each tumour location.