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Biomedical subjects

T Dalkara

Publications and source records attributed to T Dalkara.

At least 19 recordsLinked to original sources
PubMed2006

Serial analysis of gene expression in the hippocampus of patients with mesial temporal lobe epilepsy.

Hippocampal sclerosis constitutes the most frequent neuropathological finding in patients with medically intractable mesial temporal lobe epilepsy. Serial analysis of gene expression was used to get a global view of the gene profile in human hippocampus in control condition and in epileptic condition associated with hippocampal sclerosis. Libraries were generated from control hippocampus, obtained by rapid autopsy, and from hippocampal surgical specimens of patients with mesial temporal lobe epilepsy and the classical pattern of hippocampal sclerosis. More than 50,000 tags were analyzed (28,282, control hippocampus; 25,953, hippocampal sclerosis) resulting in 9206 (control hippocampus) and 9599 (hippocampal sclerosis) unique tags (genes), each representing a specific mRNA transcript. Comparison of the two libraries resulted in the identification of 143 transcripts that were differentially expressed. These genes belong to a variety of functional classes, including basic metabolism, transcription regulation, protein synthesis and degradation, signal transduction, structural proteins, regeneration and synaptic plasticity and genes of unknown identity of function. The database generated by this study provides an extensive inventory of genes expressed in human control hippocampus, identifies new high-abundant genes associated with altered hippocampal morphology in patients with mesial temporal lobe epilepsy and serves as a reference for future studies aimed at detecting hippocampal transcriptional responses under various pathological conditions.

Base Sequence↗
PubMed2006

From spreading depression to the trigeminovascular system.

Migraine headaches have a complex pathophysiology; both vascular and neuronal mechanisms have been proposed. One possible scenario begins with brain-initiated events evolving to cortical spreading depression (CSD), which in turn activates the trigeminal nerve to cause headaches. Experimental evidence supports a relationship between CSD as a cause of migraine aura as well as CSD as a cause of trigeminal activation. Susceptibility to CSD and to migraine appears to be genetically determined. In some migraine subtypes, genes controlling translocation of calcium, sodium and potassium have been implicated, perhaps altering the susceptibility to CSD. This chapter briefly reviews current knowledge pertaining to migraine pathophysiology with emphasis on current notions linking disturbances in ion flux to the genesis of headache.

Cerebrovascular Circulation↗
PubMed2005

Preparation and characterization of PLGA nanospheres for the targeted delivery of NR2B-specific antisense oligonucleotides to the NMDA receptors in the brain.

Treatment of central nervous system (CNS) diseases with potentially useful pharmaceuticals is prevented by the blood-brain barrier (BBB). The BBB is a unique protective barrier in the body. It is formed by epithelial-like tight junctions, which are expressed by the brain capillary endothelial cells. Although most molecules are potentially active in the CNS, they cannot readily enter the brain because of their properties. Antisense oligonucleotides (ODNs) have a great potential as neuropharmaceuticals; however, the large size and polar nature of nucleic acid drugs prevent these molecules from bypassing the BBB and readily entering the CNS following systemic administration. One approach to improve both the pharmacokinetics and the pharmacodynamics of ODNs involves the use of sustained-release polymer formulations, such as poly(lactide-co-glycolide) (PLGA) nanoparticulate systems. In this study, nanospheres were prepared by the emulsification diffusion technique and characterized in terms of particle size, surface morphology, encapsulation efficiency, in vitro release profiles and ODN stability. The nanospheres produced were spherical with homogenous size distribution. Nanospheres were prepared with different encapsulation efficiency. Release profiles of formulations were also evaluated. The results show that formulations with different ODN content exhibited different release profiles. Moreover, the chemical integrity of ODN during the processes was conserved. These results demonstrate that a stable ODN formulation could be prepared utilizing PLGA nanospheres as a potential delivery system for the treatment of CNS diseases.

Brain↗
PubMed2004

In vitro cytotoxicity of mitoxantrone-incorporated albumin microspheres on acute promyelocytic leukaemia cells.

In the present study, the preparation and characterization of bovine serum albumin (BSA) microspheres and the evaluation of the in vitro cytotoxicity of these microspheres on acute promyelocytic leukaemia (HL-60) cells were described. Mitoxantrone (MTZ)-incorporated microspheres were evaluated for particle size, drug loading, release characteristics and surface morphology. The biological effect of MTZ released from BSA microspheres was determined on an in vitro cultured HL-60 cell line, showing that, after encapsulation, MTZ still retains cytotoxic activity. For this purpose, methyl-thiazol-tetrazolium (MTT) assay was used to evaluate the in vitro cytotoxicity of MTZ-loaded microspheres. Particle size of BSA microspheres was determined between 17.61-20.38 microm and they were smooth and spherical in shape. Encapsulation efficiency of the drug-loaded microspheres was between 22.26-60.50%. For MTZ-containing microspheres, the cell death ratios were greater than 80% for all formulations. This study demonstrate that BSA microspheres were well suited for the controlled release of MTZ and were promising for anti-cancer chemotherapy.

Animals↗
PubMed2002

How to construct a research project.

A research proposal should start with defining its aims. A synopsis of the literature and observations that the hypothesis is grounded should be given together with relevant references. The significance of the project, expected contributions to the field should also be indicated. If available, preliminary data enhance the impact of the proposal. A detailed description of the methods and subjects to be used is an important part of the proposal. What data are to be collected, the method of collecting data, and selection criteria for subjects should be indicated. Variables and how the measurements will be taken, have to be defined precisely. Data processing and analysis tools should be described. Additionally, the expected time table, project cost, the ethical and legal issues should be included in the proposal. It should be kept in mind that a scientist's primary responsibility is to create conditions such that the hypothesis can be tested objectively rather than proving his hypothesis.

Clinical Trials as Topic↗
PubMed2001

Chronic daily administration of selegiline and EGb 761 increases brain's resistance to ischemia in mice.

Brief cerebral ischemia is reported to cause selective neuronal necrosis, apoptotic cell death, silent infarcts and, when recurrent, cognitive decline. Acute administration of selegiline and EGb 761 have been shown to have anti-apoptotic and neuroprotective effects in experimental ischemia. Their daily use is currently advised to slow down cognitive decline in patients with vascular dementia. Hence, unlike previous studies, we studied the neuroprotective action of chronic daily administration of these drugs in Swiss mice subjected to 30-min middle cerebral artery occlusion and 72 h of reperfusion since this model was reported to induce a slowly evolving infarct with profuse apoptotic cell death. Infarct area was evaluated by H&E staining on coronal brain sections and, apoptotic cells were identified by histological criteria, terminal transferase-mediated d-UTP nick-end labeling (TUNEL) and by immunohistochemical detection of caspase-cleaved actin fragments (fractin). Fifty-one mice received daily intraperitoneal injections of 10 mg/kg selegiline (n=18) or 50 mg/kg EGb 761 (n=17) or equal volume of saline (n=16) for 10-14 days before but not on the day of insult. The infarct volume, number of TUNEL- and fractin-positive cells were significantly reduced in treatment groups by 30, 42 and 51% (selegiline) and, 27, 27 and 29% (EGb 761), respectively. These data suggest that prophylactic use of selegiline and EGb 761 could increase the brain's resistance to mild ischemic injury.

Actins↗
PubMed2001

Nitric oxide is involved in ischemia-induced apoptosis in brain: a study in neuronal nitric oxide synthase null mice.

Nitric oxide can promote or inhibit apoptosis depending on the cell type and coexisting metabolic or experimental conditions. We examined the impact of nitric oxide on development of apoptosis 6, 24, and 72 h after permanent middle cerebral artery occlusion in mutant mice that lack the ability to generate nitric oxide from neuronal nitric oxide synthase. Adjacent coronal sections passing through the anterior commissure were stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Immunoblotting was used to identify changes in the anti- and proapoptotic proteins Bcl-2 and Bax, respectively. Activation of caspases was assessed by appearance of actin cleavage products using a novel antiserum directed against 32-kDa actin fragment (fractin). In the neuronal nitric oxide synthase mutant mouse, infarct size and TUNEL positive apoptotic neurons were reduced compared to the wild-type controls. At 6 h, Bcl-2 levels in the ischemic hemisphere were increased in mutants but decreased in the wild-type strain. Bax levels did not change significantly. Caspase-mediated actin cleavage appeared in the ischemic hemisphere at this time point, and was significantly less in mutant brains at 72 h compared to the wild-type. The reduction in the number of TUNEL and fractin positive apoptotic cells appears far greater than anticipated based on the smaller lesion size in mutant mice.Hence, from these data we suggest that a deficiency in neuronal nitric oxide production slows the development of apoptotic cell death after ischemic injury and is associated with preserved Bcl-2 levels and delayed activation of effector caspases.

Actins↗
PubMed2001

Synergistic protective effect of caspase inhibitors and bFGF against brain injury induced by transient focal ischaemia.

We tested the hypothesis that combined use of trophic factors and caspase inhibitors increases brain resistance to ischaemia in mice. Intracerebroventricular administration of bFGF (>10 ng) 30 min after MCA occlusion decreased infarct size and neurological deficit in a dose-dependent manner following 2 h ischemia and reperfusion (20 h). Combined administration of the subthreshold doses of bFGF (3 ng) and caspase inhibitors (z-VAD.FMK, 27 ng or z-DEVD.FMK, 80 mg) reduced infarct volume by 60%, and reduced neurological deficit. Treatment with a subthreshold dose of bFGF (3 ng) extended the therapeutic window for z-DEVD.FMK (480 ng) from 1 to 3 h after reperfusion. Caspase-3 activity in the ischaemic brain was increased 30 min and 2 h after reperfusion but, was significantly reduced in bFGF-treated animals by 29 and 16%, respectively. Caspase-3 activity was not reduced by a direct bFGF effect because addition of bFGF (10 nM - 2 microM) did not decrease recombinant caspase-3 activity, in vitro. Our data show that combining caspase inhibitors and bFGF lengthens the treatment window for the second treatment, plus lowers the dosage requirements for neuroprotection. These findings are important because low doses of caspase inhibitors or bFGF reduce the possibility of side effects plus extend the short treatment window for ischaemic stroke.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Association of nitric oxide production and apoptosis in a model of experimental nephropathy.

BACKGROUND: In recent studies increased amounts of nitric oxide (NO) and apoptosis have been implicated in various pathological conditions in the kidney. We have studied the role of NO and its association with apoptosis in an experimental model of nephrotic syndrome induced by a single injection of adriamycin (ADR). METHODS: The alteration in the NO pathway was assessed by measuring nitrite levels in serum/urine and by evaluating the changes in vascular reactivity of the isolated perfused rat kidney (IPRK) system. Rats were stratified into control groups and ADR-induced nephropathy groups. These two groups were then divided into: group 1, animals receiving saline; and group 2, animals receiving aminoguanidine (AG) which is a specific inhibitor of inducible-NO synthase. On day 21, rats were sacrificed after obtaining material for biochemical analysis. RESULTS: Histopathological examination of the kidneys of rats treated with ADR revealed focal areas of mesangial proliferation and mild tubulointerstitial inflammation. They also had significantly higher levels of proteinuria compared with control and treatment groups (P < 0.05). Urine nitrite levels were significantly increased in the ADR-nephropathy group (P < 0.05). In the IPRK phenylephrine and acetylcholine related responses were significantly impaired in the ADR-nephropathy group. Apoptosis was not detected in controls. However, in the ADR-nephropathy group, numerous apoptotic cells were identified in the tubulointerstitial areas. Double staining revealed numerous interstitial apoptotic cells to stain for ED1, a marker for monocytes/macrophages. Treatment with AG prevented the impairment of renal vascular bed responses and reduced both urine nitrite levels and apoptosis to control levels. CONCLUSION: We suggest that interactions between NO and apoptosis are important in the pathogenesis of the ADR-induced nephrosis.

Animals↗
PubMed2001

Spleen damage in endotoxaemic mice: the involvement of nitric oxide.

The association between Escherichia coli endotoxin-induced organ damage and nitric oxide-related mechanisms was investigated in the spleen of male Swiss albino mice (20-40 g) by using (1) Pt/Ir electrochemical sensor connected to an amperometric detection system (NO-501, InterMedical Co., Japan), (2) nitrotyrosine immunohistochemistry, (3) conventional light microscopy and (4) immunoblotting techniques in parallel. 1 h before endotoxin injection, animals were pretreated with either nitric oxide synthase inhibitor, L-N(G)-nitroarginine methyl ester (L-NAME, 20 mg kg(-1), i.p.) or inducible nitric oxide synthase expression inhibitor, dexamethasone (5 mg kg(-1), i.p.) or the inhibitor of murine inducible nitric oxide synthase in vivo, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT, 1 mg kg(-1), i.p.). 5 h after endotoxin treatment, electrochemically detected concentration of nitric oxide was significantly elevated (nM, endotoxin: 716.6 +/- 178.2, n = 10 vs saline: 209.4 +/- 127.8, n = 9, P = 0.0312, unpaired Student's t-test) and remained so throughout the 30 min monitorization period. Neither dexamethasone nor AMT blocked the endotoxin-induced overproduction of nitric oxide indicating that the enhanced inducible nitric oxide synthase activity cannot be the only explanation. When dexamethasone and L-NAME combination was used to block both the constitutive and the inducible isoforms, nitric oxide production was virtually abolished, indicating a significant contribution from the constitutive isoform of nitric oxide synthase. The results of nitrotyrosine immunohistochemistry and the conventional light microscopy were also in agreement with the amperometric method while immunoblotting revealed the expression of both the endothelial and the inducible isoforms of nitric oxide synthase were induced endotoxaemic animals. Thus, conclude that endotoxin-induced splenic damage in endotoxaemia can be explained by enhanced production of nitric oxide due to the induction of both endothelial and inducible nitric oxide synthases while causal relationship and the roles of other deleterious mediators such as oxygen-derived free radicals are yet to be established.

Animals↗
PubMed2000

Altered mechanisms of motor-evoked potential generation after transient focal cerebral ischemia in the rat: implications for transcranial magnetic stimulation.

We recently demonstrated that a long-lasting transmission defect in cortical synapses caused motor dysfunction after brief middle cerebral artery (MCA) occlusion in the rat despite rapid recovery of axons. In this experimental study, we have examined the impact of differential recovery of synapses and axons on generation of motor-evoked potentials (MEP) recorded from contralateral paralyzed and ipsilateral unaffected muscles, to gain insight into mechanisms of MEPs recorded from stroke patients by transcranial magnetic stimulation (TMS). MEPs generated by focal electrical stimulation of the forelimb area of motor cortex were simultaneously recorded from the brain stem, contra- and ipsilateral forelimb and contralateral hindlimb muscles in rats subjected to transient MCA occlusion. The effect of ischemia on cortical activity and axonal conduction was differentially studied by proximal or distal occlusion of the MCA. Regional cerebral blood flow changes in the forelimb area were monitored by laser-Doppler flowmetry during ischemia and reperfusion. In addition, synaptic transmission within the forelimb area of motor cortex was examined by intracellular and extracellular recording of potentials generated by stimulation of the premotor area. No MEP response was recorded during ischemia. Upon reperfusion: (i) motor axons readily regained their excitability and cortical stimulation caused successive pyramidal volleys (recorded as D waves from the brain stem) and a MEP from contralateral paralytic muscles although synaptic activation of motor pathways was not feasible; (ii) the amplitude of pyramidal volley was increased; (iii) MEPs with a longer latency were recorded from the ipsilateral forelimb. In conclusion, differential recovery of synapses and axons after ischemia may account for some previously unexplained findings (such as preserved MEPs in paralysed muscles) observed in cortical stimulation studies of stroke patients.

Animals↗
PubMed2000

Endothelial nitric oxide synthase-dependent cerebral blood flow augmentation by L-arginine after chronic statin treatment.

Nitric oxide, a product of nitric oxide synthase activity, relaxes vascular smooth muscle and elevates brain blood flow. We evaluated the importance of eNOS to cerebral blood flow augmentation after L-arginine infusion and increases in flow after eNOS upregulation in SV-129 mice. Blood flow was measured by laser-Doppler flowmetry before and after L-arginine infusion (450 mg/kg during a 15-minute period) or measured by 14C-iodoamphetamine indicator fractionation or 14C-iodoantipyrine tissue equilibration techniques. rCBF increased by 26% (laser Doppler flowmetry) after L-arginine infusion but did not change in mutant mice deficient in eNOS expression. After eNOS upregulation by chronic simvastatin treatment (2 mg/kg subcutaneously, daily for 14 days), L-arginine amplified and sustained the hyperemia (38%) and increased absolute brain blood flow from 86 +/- 7 to 119 +/- 10 mL/100 g per minute. Furthermore, pretreatment with simvastatin enhanced blood flow within ischemic brain tissue after middle cerebral artery occlusion. Together, these findings suggest that eNOS activity is critical for blood flow augmentation during acute L-arginine infusion, and chronic eNOS upregulation combined with L-arginine administration provides a novel strategy to elevate cerebral blood flow in the normal and ischemic brain.

Animals↗
PubMed2000

Role of endothelial nitric oxide generation and peroxynitrite formation in reperfusion injury after focal cerebral ischemia.

BACKGROUND AND PURPOSE: Reperfusion injury is one of the factors that unfavorably affects stroke outcome and shortens the window of opportunity for thrombolysis. Surges of nitric oxide (NO) and superoxide generation on reperfusion have been demonstrated. Concomitant generation of these radicals can lead to formation of the strong oxidant peroxynitrite during reperfusion. METHODS: We have examined the role of NO generation and peroxynitrite formation on reperfusion injury in a mouse model of middle cerebral artery occlusion (2 hours) and reperfusion (22 hours). The infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride staining; blood-brain barrier permeability was evaluated by Evans blue extravasation. Nitrotyrosine formation and matrix metalloproteinase-9 expression were detected by immunohistochemistry. RESULTS: Infarct volume was significantly decreased (47%) in animals treated with the nonselective nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NA) at reperfusion. The specific inhibitor of neuronal NOS, 7-nitroindazole (7-NI), given at reperfusion, showed no protection, although preischemic treatment with 7-NI decreased infarct volume by 40%. Interestingly, prereperfusion administration of both NOS inhibitors decreased tyrosine nitration (a marker of peroxynitrite toxicity) in the ischemic area. L-NA treatment also significantly reduced vascular damage, as indicated by decreased Evans blue extravasation and matrix metalloproteinase-9 expression. CONCLUSIONS: These data support the hypothesis that in addition to the detrimental action of NO formed by neuronal NOS during ischemia, NO generation at reperfusion plays a significant role in reperfusion injury, possibly through peroxynitrite formation. Contrary to L-NA, failure of 7-NI to protect against reperfusion injury suggests that the source of NO is the cerebrovascular compartment.

Animals↗
PubMed2000

In vitro/in vivo studies on a buccal bioadhesive tablet formulation of carbamazepine.

A buccoadhesive controlled-release system for delivery of carbamazepine (CBZ) was prepared by compression of hydroxypropyl methylcellulose (HPMC) and carbomer, incorporating a penetration enhancer, sodium glycodeoxycholate (GDC). The release behaviour of systems containing CBZ and various amounts of the two polymers with and without GDC was found to be non-Fickian. Formation of an interpolymer complex between HPMC and carbomer was confirmed in acidic medium by turbidity, viscosity and FT-IR measurements. Addition of the drug to the buccoadhesive formulation reduced the adhesion force significantly (p < 0.1). GDC did not have any effect on bioadhesion. Permeability of bovine buccal mucosa to CBZ was determined using Ussing diffusion chambers [1]. In vivo interaction between the tablet and tissue was examined histologically as well as by scoring mucosal irritation. Histological changes observed in the buccal epithelium after 4 h contact with the tablets containing GDC recovered completely within 24 h after removal. No measurable plasma level of CBZ was obtained either in the absence or presence of GDC.

Absorption↗
PubMed1999

Compartmental changes in expression of c-Fos and FosB proteins in intact and dopamine-depleted striatum after chronic apomorphine treatment.

Chronic administration of dopaminergic agonists to rats with unilateral 6-OH-dopamine (6-OHDA) lesions of nigrostriatal pathway produces behavioral sensitization to subsequent agonist challenges and may serve as a model for DOPA-induced dyskinesias. In order to understand striatal mechanisms behind this long-term behavioral change we examined striatal c-Fos and FosB immunoreactivity induced by apomorphine challenge (5 mg/kg, s.c.) after 3 days of withdrawal following a 2-week administration (5 mg/kg, b.i.d., s.c.) both in intact and 6-OHDA-lesioned animals. In intact rats, c-Fos induction by acute apomorphine exposure showed a striosomal pattern, whereas FosB immunopositivity was diffusely distributed. Following chronic administration, FosB induction turned to a clear striosome dominant pattern similar to c-Fos expression. In denervated striatum, expression of both proteins was profoundly augmented in a homogeneous pattern after a single dose of apomorphine. A distinct striosomal patterning appeared after chronic apomorphine administration in ventromedial part of the denervated striatum with a down-regulation in the matrix and relative enhancement in striosomes. These results suggest that compartmental reorganization of striatal neuronal activity may play a role in long-term behavioral changes induced by chronic dopaminergic treatments both under normal and dopamine-depleted conditions.

Animals↗
PubMed1999

Occlusion of the MCA by an intraluminal filament may cause disturbances in the hippocampal blood flow due to anomalies of circle of Willis and filament thickness.

We examined blood flow changes and histology in the hippocampus induced by occlusion of the middle cerebral artery (MCA) by a filament in Swiss albino and SV-129 mice (n=67) and in Wistar rats (n=64). Filling cerebral arteries with carbon black revealed that one or both posterior communicating arteries were hypoplastic in 50% of Swiss mice. Ischemic changes were detected in the ipsilateral hippocampus with 2,3,5-triphenyl tetrazolium chloride or hematoxylin and eosin staining when these mice were subjected to 2-h MCA occlusion and 22-h reperfusion. No such abnormalities were found in SV-129 mice and Wistar rats (except one). The hippocampal blood flow dropped to 60+/-2.3% of the baseline in mice with a normal circle of Willis but to 37+/-4.2% in those with an incomplete circle when the MCA was occluded with a 6/0 nylon filament. When an 8/0 filament was used, no flow change in mice with a normal circle but a decrease to 60+/-2% in those with an incomplete circle was observed. A flow drop to 63+/-4% was also seen in Wistar rats when a 3/0 filament used. These data demonstrate that occlusion of the MCA by a thick filament may cause flow reduction in the hippocampus, which may be severe enough to lead to infarction if the circle of Willis is anomalous.

Animals↗
PubMed1999

Apoptosis in brain biopsies of subacute sclerosing panencephalitis patients.

Subacute sclerosing panencephalitis (SSPE) is associated with inflammatory infiltration, neuronal loss, and demyelination. The pathogenesis of these changes is unclear. We examined DNA fragmentation and Bcl-2 expression in brain biopsies of nineteen SSPE patients to investigate the role of apoptosis in tissue damage. DNA fragmentation was present in oligodendroglia, and, in tissues with neuronal loss, in neurons. Reactive astrocytes had no DNA fragmentation, but strong Bcl-2 expression. These results suggest apoptosis as one of the mechanisms for oligodendroglial and neuronal death in SSPE.

Adolescent↗