Biomedical subjects

R Busto

Publications and source records attributed to R Busto.

At least 19 recordsLinked to original sources

PubMed2003

Hyperbaric oxygen therapy protects against mitochondrial dysfunction and delays onset of motor neuron disease in Wobbler mice.

The Wobbler mouse is a model of human motor neuron disease. Recently we reported the impairment of mitochondrial complex IV in Wobbler mouse CNS, including motor cortex and spinal cord. The present study was designed to test the effect of hyperbaric oxygen therapy (HBOT) on (1) mitochondrial functions in young Wobbler mice, and (2) the onset and progression of the disease with aging. HBOT was carried out at 2 atmospheres absolute (2 ATA) oxygen for 1 h/day for 30 days. Control groups consisted of both untreated Wobbler mice and non-diseased Wobbler mice. The rate of respiration for complex IV in mitochondria isolated from motor cortex was improved by 40% (P<0.05) after HBOT. The onset and progression of the disease in the Wobbler mice was studied using litters of pups from proven heterozygous breeding pairs, which were treated from birth with 2 ATA HBOT for 1 h/day 6 days a week for the animals' lifetime. A "blinded" observer examined the onset and progression of the Wobbler phenotype, including walking capabilities ranging from normal walking to jaw walking (unable to use forepaws), and the paw condition (from normal to curled wrists and forelimb fixed to the chest). These data indicate that the onset of disease in untreated Wobbler mice averaged 36+/-4.3 days in terms of walking and 40+/-5.7 days in terms of paw condition. HBOT significantly delayed (P<0.001 for both paw condition and walking) the onset of disease to 59+/-8.2 days (in terms of walking) and 63+/-7.6 days (in terms of paw condition). Our data suggest that HBOT significantly ameliorates mitochondrial dysfunction in the motor cortex and spinal cord and greatly delays the onset of the disease in an animal model of motor neuron disease.

Animals↗

PubMed2002

Expression of mRNA for growth hormone-releasing hormone and splice variants of GHRH receptors in human malignant bone tumors.

Splice variants (SV) of receptors for growth hormone-releasing hormone (GHRH) have been found in several human cancer cell lines. GHRH antagonists inhibit growth of various human cancers, including osteosarcomas and Ewing's sarcoma, xenografted into nude mice or cultured in vitro and their antiproliferative action could be mediated, in part, through these SV of GHRH receptors. In this study, we found mRNA for the SV(1) isoform of GHRH receptors in human osteosarcoma line MNNG/HOS and SK-ES-1 Ewing's sarcoma line. We also detected mRNA for GHRH, which is apparently translated into the GHRH peptide and secreted by the cells, as shown by the presence of GHRH-like immunoreactivity in the conditioned media of cell cultures. In proliferation studies in vitro, the growth of SK-ES-1 and MNNG/HOS cells was dose-dependently inhibited by GHRH antagonist JV-1-38 and an antiserum against human GHRH. Our study indicates the presence of an autocrine stimulatory loop based on GHRH and SV(1) of GHRH receptors in human sarcomas. The direct antiproliferative effects of GHRH antagonists on malignant bone tumors appear to be exerted through the SV(1) of GHRH receptors on tumoral cells.

Alternative Splicing↗

PubMed2002

Targeted cytotoxic analogue of luteinizing hormone-releasing hormone (LH-RH) only transiently decreases the gene expression of pituitary receptors for LH-RH.

A cytotoxic analogue of LH-RH, AN-207, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier [D-Lys6]LH-RH, was developed for targeted therapy of cancers expressing LH-RH-receptors. To determine its possible side-effects on the pituitary gland, we investigated the gene expression of pituitary LH-RH-receptors and LH secretion in ovariectomized female and normal male rats after treatment with the maximum tolerated dose of AN-207. The effect of AN-207 on the gene expression of the pituitary GH-RH-receptors and GH secretion was also assessed in male rats. Five hours after a single i.v. injection of AN-207 at 175 nmol/kg, there was a 39-51% decrease in mRNA expression for the pituitary LH-RH-receptors in male and female rats. The carrier, at an equimolar dose, caused a similar reduction (37-39%), whereas the cytotoxic radical AN-201, at an equitoxic dose (110 nmol/kg), produced only a 12-24% decrease (NS) in the mRNA expression of LH-RH-receptors. AN-207 and the carrier analogue induced a comparable 90-100-fold increase in serum LH concentrations in male rats, and the same 12-fold elevation in OVX rats at 5 h. Seven days after treatment with AN-207, the mRNA levels for the LH-RH receptors and the serum LH concentration were back to normal in both sexes. AN-207, the carrier, and AN-201 had no significant effect on the expression of mRNA for GH-RH-receptors in the pituitary. In vitro, a continuous perfusion of pituitary cells with 10 nM AN-207 did not affect the hormone-releasing function of the targeted LH cells or the nontargeted GH cells. Our results demonstrate that cytotoxic LH-RH analogue AN-207, at the maximum tolerated dose causes only a transient decrease in the gene expression of the pituitary LH-RH receptors, and the levels of mRNA for LH-RH receptor fully recover within 7 days. Moreover, the carrier hormone moiety, and not the cytotoxic radical in AN-207 is responsible for this transient suppression. Our findings suggest that the therapy with cytotoxic LH-RH analogues will not inflict permanent damage to pituitary function.

Animals↗

PubMed2002

Inhibitory effects of antagonistic analogs of GHRH on GH3 pituitary cells overexpressing the human GHRH receptor.

GH3 rat pituitary tumor cells produce GH and prolactin (PRL), but lack the GHRH receptor (GHRH-R). We expressed human GHRH-R (hGHRH-R) in GH3 cells using recombinant adenoviral vectors and studied the effects of GHRH antagonists. The mRNA expression of the GHRH-R gene in the cells was demonstrated by RT-PCR. An exposure of the GH3 cells infected with hGHRH-R to 10(-10), 10(-9) and 10(-8) m hGHRH for 1 or 2 h in culture caused a dose-dependent elevation of the intracellular cAMP concentration and the cAMP efflux. Exposure to hGHRH also elicited dose-dependent increases in GH and PRL secretion from these cells. Neither the uninfected nor the antisense hGHRH-R-infected control cells exhibited cAMP, GH and PRL responses to GHRH stimulation. GHRH antagonists JV-1-38 and jv-1-36 applied at 3x10(-8) m for 3 h, together with 10(-9) m GHRH, significantly inhibited the GHRH-stimulated cAMP efflux from the hGHRH-R-infected cells by 36 and 80% respectively. The more potent antagonist JV-1-36 also decreased the intracellular cAMP levels in these cells by 55%. Exposure to JV-1-36 for 1 h nullified the stimulatory effect of GHRH on GH secretion and significantly inhibited it by 64 and 77% after 2 and 3 h respectively. In a superfusion system, GHRH at 10(-10), 10(-9) and 10(-8) m concentrations induced prompt and dose-related high cAMP responses and smaller increases in the spontaneous GH secretion of the hGHRH-R-infected cells. Antagonists JV-1-36 and JV-1-38 applied at 3x10(-8) m for 15 min, together with 10(-9) m GHRH, inhibited the GHRH-stimulated cAMP response by 59 and 35% respectively. This work demonstrates that GHRH antagonists can effectively inhibit the actions of GHRH on the hGHRH-R. Our results support the view that this class of compounds would be active clinically.

Animals↗

PubMed2001

Neuroprotective effect of treatment with human albumin in permanent focal cerebral ischemia: histopathology and cortical perfusion studies.

In recent experimental studies, we demonstrated a highly beneficial neuroprotective effect of moderate- to high-dose human albumin treatment of transient focal cerebral ischemia, but we did not define the effect of albumin therapy in permanent focal cerebral ischemia. In this study, anesthetized Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion by retrograde insertion of an intraluminal nylon suture coated with poly-L-lysine. Albumin was administered i.v. at 2 h after onset of middle cerebral artery occlusion, in doses of either 1.25 (n=8) or 2.5 g/kg (n=6). In a separate group of animals, albumin (2.5 g/kg) was given 1 h after middle cerebral artery occlusion (n=6). Vehicle-treated rats (n=6) received 0.9% saline in equivalent volumes. Neurological status was evaluated during and 24 h after middle cerebral artery occlusion. One day after middle cerebral artery occlusion, infarct volumes and brain edema were determined. In a separate group of animals, cortical perfusion was assessed by Laser-Doppler perfusion imaging. Albumin (1.25 g/kg; n=3) or vehicle (sodium chloride 0.9%; n=3) was administered at 2 h after onset of middle cerebral artery occlusion. Higher-dose albumin therapy (2.5 g/kg) significantly improved the neurological score compared to vehicle rats at 24 h, when administered at either 1 or 2 h after middle cerebral artery occlusion. Total infarct volume was reduced by albumin (2.5 g/kg given at 2 h) by 32% compared with vehicle-treated rats. Both albumin doses (1.25 and 2.5 g/kg) significantly reduced cortical and striatal infarct areas at several coronal levels when administered at 2 h after middle cerebral artery occlusion. Brain swelling was not affected by albumin treatment. Cortical perfusion declined during middle cerebral artery occlusion in both groups. Treatment with albumin led to 48% increases in cortical perfusion (P<0.002), but saline caused no change. These results support a beneficial effect of albumin therapy in permanent focal cerebral ischemia.

Animals↗

PubMed2001

Transient changes of brain-derived neurotrophic factor (BDNF) mRNA expression in hippocampus during moderate ischemia induced by chronic bilateral common carotid artery occlusions in the rat.

Chronic bilateral common carotid artery occlusion (BCCAO) induces moderate ischemia (oligemia) in the rat forebrain in the absence of overt neuronal damage. In situ hybridization for brain-derived neurotrophic factor (BDNF) mRNA was used to search for a molecular response to moderate ischemia. BDNF mRNA was significantly increased in the hippocampal granule cells at 6 h of occlusion (ANOVA, Tukey test P<0.05). At 1, 7 and 14 days BDNF mRNA levels returned to control levels. The frequency of BDNF gene expression at 6 h was 83%, which was significantly higher than the 7% incidence of histological injury in the hippocampus (Fisher's exact test, P<0.002). Cerebral blood flow was reduced to 75% of control levels in the hippocampus after 1 week of BCCAO when measured with the autoradiographic method. Measurements of tissue flow with a microprobe for laser Doppler flow excluded decreases into the ischemic range during the period when elevated gene expression was observed. Prolonged moderate ischemia (oligemia) is a sufficient stimulus for BDNF gene expression in the hippocampus. These molecular studies provide direct evidence for an involvement of the hippocampus in the BCCAO model.

Animals↗

PubMed2001

Simultaneous measurement of cerebral blood flow and mRNA signals: pixel-based inter-modality correlational analysis.

The analysis of pixel-based relationships between local cerebral blood flow (LCBF) and mRNA expression can reveal important insights into brain function. Traditionally, LCBF and in situ hybridization studies for genes of interest have been analyzed in separate series. To overcome this limitation and to increase the power of statistical analysis, this study focused on developing a double-label method to measure local cerebral blood flow (LCBF) and gene expressions simultaneously by means of a dual-autoradiography procedure. A 14C-iodoantipyrine autoradiographic LCBF study was first performed. Serial brain sections (12 in this study) were obtained at multiple coronal levels and were processed in the conventional manner to yield quantitative LCBF images. Two replicate sections at each bregma level were then used for in situ hybridization. To eliminate the 14C-iodoantipyrine from these sections, a chloroform-washout procedure was first performed. The sections were then processed for in situ hybridization autoradiography for the probes of interest. This method was tested in Wistar rats subjected to 12 min of global forebrain ischemia by two-vessel occlusion plus hypotension, followed by 2 or 6 h of reperfusion (n=4-6 per group). LCBF and in situ hybridization images for heat shock protein 70 (HSP70) were generated for each rat, aligned by disparity analysis, and analyzed on a pixel-by-pixel basis. This method yielded detailed inter-modality correlation between LCBF and HSP70 mRNA expressions. The advantages of this method include reducing the number of experimental animals by one-half; and providing accurate pixel-based correlations between different modalities in the same animals, thus enabling paired statistical analyses. This method can be extended to permit correlation of LCBF with the expression of multiple genes of interest.

Algorithms↗

PubMed2001

The effect of bone morphogenetic protein-7 (BMP-7) on functional recovery, local cerebral glucose utilization and blood flow after transient focal cerebral ischemia in rats.

Bone morphogenetic protein-7 (BMP-7) has been shown to enhance dendritic growth and improve functional recovery after experimental stroke. In this study, we examined the effect of BMP-7 on functional recovery, local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCMRglu) following transient middle cerebral artery occlusion. Sprague--Dawley rats (n=29) were anesthetized with halothane/nitrous oxide and received 2-h middle cerebral artery occlusion (MCAo) by poly-L-lysine-coated intraluminal suture. Rectal and cranial temperatures were regulated at 37.0--37.5 degrees C. BMP-7 or vehicle (volume, 25 microl) was administered intracisternally in a blinded fashion at 24 h after MCAo. Neurological status was evaluated during occlusion (60 min) and daily for 2 days after MCAo. In matched animal groups, LCMRglu was measured autoradiographically with [(14)C]2-deoxyglucose (2-DG) and LCBF with [(14)C]iodoantipyrine 48 h after MCAo. Four animals groups were studied: LCMRglu series (BMP-7, n=7; vehicle, n=8); LCBF series (BMP-7, n=6; vehicle, n=8). Average three-dimensional image data sets were constructed for each group and were compared by pixel-based statistical methods. Rectal and cranial temperatures, mean blood pressure, plasma glucose and blood gases were similar among groups. BMP-7 significantly improved the total neurological score compared to vehicle at 48 h after MCAo (7.3+/-0.4 vs. 9.0+/-0.2, respectively; P<0.0003). Compared to vehicle-rats, BMP-7 enhanced glucose utilization in the basal ganglia ipsilateral to stroke and improved LCBF in ipsilateral subthalamus, but decreased LCBF and LCMRglu in contralateral cortical regions.

Animals↗

PubMed2001

Dysfunctional mitochondrial respiration in the wobbler mouse brain.

The involvement of mitochondrial dysfunction promoting neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), has been suggested. Histopathological and biochemical mitochondrial abnormalities have been reported in both sporadic and familial patients and suggest the contention that mitochondria may play a key role promoting ALS. Animal models of ALS provide a unique opportunity to study this incurable and fatal human disease. In the present study we tested the hypothesis that alterations in mitochondrial physiology occur in the brain of wobbler mice. No significant difference was found in the respiratory control index or adenosine diphosphate/oxygen ratio values between isolated mitochondria of wobbler and control mice. When pyruvate and malate were used as substrates, oxygen consumption was decreased significantly by approximately 33% in mitochondria isolated from wobbler mouse brain compared to controls. Oxygen consumption in the presence of ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) was decreased significantly by approximately 21% in wobbler brain mitochondria compared to controls, which suggests impairment in the function of complex IV. These findings are the first demonstration of mitochondrial respiratory chain dysfunction in the brain of the wobbler mouse.

Amyotrophic Lateral Sclerosis↗

PubMed2001

Intraischemic but not postischemic hypothermia prevents non-selective hippocampal downregulation of AMPA and NMDA receptor gene expression after global ischemia.

Hypothermia may afford histological neuroprotection induced by ischemia by preventing aberrant Ca2+ influx through NMDA (N-methyl-D-aspartic acid) or Ca2+-permeable AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors. Expression of hippocampal GluR1A, GluR2B, GluR3C and NMDAR1 (NR1) subunits was investigated by in situ hybridization at 1 and 7 days after 10-min transient global ischemia in the presence and absence of intraischemic or postischemic brain hypothermia (30 degrees C). At 1 day, normothermic ischemia markedly suppressed the expression of GluR1A, GluR2B, and GluR3C receptor mRNAs to a similar degree in the vulnerable CA1. Less vulnerable CA3a-c subregions were also acutely downregulated. NR1 mRNA expression was reduced in CA1 but to a lesser extent than AMPA mRNAs. At 7 days after normothermic ischemia, a time of marked CA1 cell loss, all three AMPA transcripts were nearly absent in CA1 while a percentage (33.9+/-7.2%) of NR1 mRNA remained. Intraischemic hypothermia fully blocked the damage and non-selective mRNA downregulations at 1 and 7 days. By contrast, postischemic hypothermia postponed neurodegeneration but only partially rescued the expression of AMPA and NR1 mRNAs at 7 days and not at 1 day after the insult. Therefore, hippocampal AMPA receptor mRNAs decline at a relatively similar rate after normothermic global ischemia and cellular neuroprotection by intraischemic hypothermia occurred independently of altered subunit composition of AMPA receptors. Since decreases persist within resistant neurons under the postischemic condition, AMPA receptor-mediated Ca2+ currents probably do not contribute to selective vulnerability.

Animals↗

PubMed2001

Hyperglycemic but not normoglycemic global ischemia induces marked early intraneuronal expression of beta-amyloid precursor protein.

Preischemic hyperglycemia is known to accentuate acute ischemic injury to neurons, microglia, and endothelia. In the present study, we used a monoclonal antibody to the N-terminal portion of beta-APP to examine how the immunoreactivity of this normal membrane glycoprotein is differentially influenced by transient cerebral ischemia when carried out under normoglycemic vs. hyperglycemic conditions. Anesthetized, physiologically regulated rats received 12.5 min of global forebrain ischemia by bilateral carotid artery occlusions plus systemic hypotension. Hyperglycemia was induced by intraperitoneal dextrose administration prior to ischemia. One or three days later, brains were examined by beta-APP immunohistochemistry. Ischemia under hyperglycemic conditions led to the robust, widespread intraneuronal expression of beta-APP immunoreactivity in neocortex, hippocampus, thalamus, and striatum of all 11 rats; this was most prominent at 24 h postischemia. Compared to rats with normoglycemic ischemia, numbers of beta-APP-immunopositive neurons in the parietal cortex of hyperglycemic rats were increased by 5.9 fold at 24 h, and by 10.6 fold at 3 days postischemia. beta-APP-immunopositive neurons in hyperglycemic rats often exhibited striking morphological alterations typical of ischemic necrosis; however, no beta-APP immunoreaction was observed in zones of frank infarction. Brains of normoglycemic rats (n=11), by contrast, showed only weak beta-APP immunostaining in occasional non-necrotic pyramidal neurons of parietal neocortex; no necrosis was present in thalamus. In sham-operated hyperglycemic rats, beta-APP immunostaining of thalamic neurons was somewhat increased at 24 h. Western analysis revealed that the hyperglycemia-induced intraneuronal overexpression of beta-APP was not associated with an overall increase in tissue levels. The results of this study demonstrate that transient forebrain ischemia under hyperglycemic conditions leads to the early intraneuronal expression of beta-APP within neuronal populations showing a heightened susceptibility to hyperglycemia-induced accentuation of ischemic injury. Our data suggest that beta-APP or its metabolites may be involved in the injury process.

Amyloid beta-Protein Precursor↗

PubMed2001

Isoform-specific membrane translocation of protein kinase C after ischemic preconditioning.

Mild cerebral anoxic/ischemic/stress insults promote 'tolerance' and thereby protect the brain from subsequent 'lethal' anoxic/ischemic insults. We examined whether specific activation of PKC alpha, delta, epsilon, or zeta isoforms is associated with ischemic preconditioning (IPC) in rat brain. IPC was produced by a 2-minute global cerebral ischemia. Membrane and cytosolic fractions of the hippocampi were immunoblotted using specific antibodies for PKCalpha, delta, epsilon, and zeta. PKCalpha showed a significant translocation to the membrane fraction from 30 min to 4 h and PKCdelta at 4 h following IPC. In contrast, the membrane/cytosol ratio of PKCepsilon showed a tendency to decrease at 30 min and 8 h, and the membrane/cytosol ratio of PKCzeta was significantly decreased from 30 min to 24 h following IPC. These findings indicate PKC isoform-specific membrane translocations in the hippocampus after brief global brain ischemia and suggest that activation of PKCalpha and PKCdelta may be associated with IPC-induced tolerance in the rat hippocampus.

Animals↗

PubMed2001

Talampanel, a novel noncompetitive AMPA antagonist, is neuroprotective after traumatic brain injury in rats.

Talampanel [(R)-7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo[4,5-h][2,3] benzodiazepine] is an orally active noncompetitive antagonist of the AMPA subtype of glutamate excitatory amino acid receptors. The purpose of this study was to determine whether treatment with talampanel would protect in a rat model of traumatic brain injury (TBI). Twenty-four hours prior to TBI, a fluid-percussion interface was positioned parasagittally over the right cerebral cortex. On the following day, fasted rats were anesthetized with 3% halothane, 70% nitrous oxide, and a balance of oxygen; mechanically ventilated and physiologically regulated; and subjected to right parieto-occipital parasagittal fluid-percussion injury (1.5-2.0 atm). The agent (talampanel, bolus infusion of 4 mg/kg followed by infusion of 4 mg/kg/h over 72 h) or vehicle was administered i.v. starting at either 30 min or 3 h after trauma. Seven days after TBI, brains were perfusion-fixed, coronal sections at various levels were digitized, and contusion areas were measured. Treatment with talampanel, when instituted 30 min after trauma, significantly reduced total contusion area compared to vehicle-treated rats (0.54 +/- 0.25 vs. 1.79 +/- 0.42 mm2, respectively). When talampanel treatment was begun at 3 h, the neuroprotective effect of the drug was lost. In addition, treatment with talampanel starting at 30 min significantly attenuated neuronal damage in all three subsectors of the hippocampal CA1 sector compared to vehicle-treated rats (normal-neuron counts, right (ipsilateral) medial CA1: 80.3 +/- 2.0 [talampanel] vs. 66.3 +/- 2.1 [vehicle] (mean +/- SEM); middle CA1: 71.5 +/- 2.0 vs. 60.3 +/- 2.2; lateral CA1: 74.5 +/- 3.0 vs. 63.0 +/- 3.2, respectively). By contrast, when talampanel treatment was begun at 3 h, normal pyramidal-neuron counts were almost identical in both groups. Our findings document that talampanel therapy instituted 30 min after trauma significantly reduces histological damage.

Animals↗

PubMed2001

Quantitative analysis of microvascular alterations in traumatic brain injury by endothelial barrier antigen immunohistochemistry.

Endothelial barrier antigen (EBA) is a protein triplet located in the plasma membrane of microvascular endothelium and selectively expressed in the normal nervous system. In this study, microvascular alterations following traumatic brain injury were studied using EBA immunohistochemistry. Anesthetized, physiologically regulated, normothermic Sprague-Dawley rats received moderate (1.5-2.0 atm) parieto-occipital parasagittal fluid-percussion traumatic brain injury (TBI). Control rats were subjected to similar anesthesia and physiological monitoring. Seven days after operative procedures, brains were perfusion-fixed, and coronal sections were reacted for EBA immunohistochemistry using a monoclonal antibody to rat EBA. Selected sections were reacted for isolectin B4 histochemistry. Computerized image analysis was used to compute numbers of EBA-immunopositive vascular profiles and mean vascular profile areas. In control brains, virtually all brain microvessels were clearly and positively immunostained, and antibody binding was specific for blood vessels. In rats with TBI, EBA immunoreactivity was greatly reduced in the zone of cortical contusion. Within the core contusion, fractional areas occupied by vascular profiles were markedly reduced (on average, by 57%), vascular profile counts were diminished, and lectin histochemistry revealed a robust inflammatory response with abundant macrophages. Taken together, these findings were thought to indicate frank microvascular destruction. At adjacent peri-contusional sites, the intensity of EBA immunostaining was also diminished; and vascular profile counts were reduced at adjacent cortical sites and homologous contralateral sites. The latter findings were interpreted as sublethal microvascular alterations possibly related to cerebral edema. The present results confirm that EBA is a specific immunohistochemical marker of normal central nervous system microvessels; that it is suitable for use in formaldehyde-fixed material; and that it is useful in quantitatively assessing microvascular alterations observed at contusional, peri-contusional and more remote sites following traumatic brain injury.

Animals↗

PubMed2001

Inhibition of growth and metastases of MDA-MB-435 human estrogen-independent breast cancers by an antagonist of growth hormone-releasing hormone.

Antagonists of growth hormone-releasing hormone (GH-RH) inhibit the growth of various cancers by mechanism(s) that include the suppression of the insulin-like growth factors (IGF)-I and/or -II. In this study, nude mice bearing orthotopic implants of MDA-MB-435 human estrogen-independent breast carcinoma received 39 days of therapy with GH-RH antagonist JV-1-36 (20 microg/day). The treatment significantly inhibited tumor growth by 71.1% (p<0.01) and nullified the metastatic potential of MDA-MB-435 cells. Four of eight control mice (50%) developed metastases in the lymph nodes and one (12.5%) in the lung, but none of the animals receiving JV-1-36 showed metastatic spread. GH-RH antagonist JV-1-36 inhibited the growth of MDA-MB-435 cells in vitro, while IGF-I stimulated it. However, mRNA for IGF-I or -II was not detected in MDA-MB-435 cells, indicating that the suppression of autocrine IGFs may not be involved in the antiproliferative mechanism. Using ligand competition assays with (125)I-labeled GH-RH antagonist JV-1-42, specific high-affinity binding sites for GH-RH were found on tumor membranes. Reverse transcription-polymerase chain reaction revealed the expression of mRNA for GH-RH receptor splice variant-1 in MDA-MB-435 tumors. Our results suggest that the antitumorigenic action of GH-RH antagonists on MDA-MB-435 breast cancer could be direct and mediated by tumoral GH-RH receptors.

Animals↗

PubMed2001

Protein aggregation after focal brain ischemia and reperfusion.

Two hours of transient focal brain ischemia causes acute neuronal death in the striatal core region and a somewhat more delayed type of neuronal death in neocortex. The objective of the current study was to investigate protein aggregation and neuronal death after focal brain ischemia in rats. Brain ischemia was induced by 2 hours of middle cerebral artery occlusion. Protein aggregation was analyzed by electron microscopy, laser-scanning confocal microscopy, and Western blotting. Two hours of focal brain ischemia induced protein aggregation in ischemic neocortical neurons at 1 hour of reperfusion, and protein aggregation persisted until neuronal death at 24 hours of reperfusion. Protein aggregates were found in the neuronal soma, dendrites, and axons, and they were associated with intracellular membranous structures during the postischemic phase. High-resolution confocal microscopy showed that clumped protein aggregates surrounding nuclei and along dendrites were formed after brain ischemia. On Western blots, ubiquitinated proteins (ubi-proteins) were dramatically increased in neocortical tissues in the postischemic phase. The ubi-proteins were Triton-insoluble, indicating that they might be irreversibly aggregated. The formation of ubi-protein aggregates after ischemia correlated well with the observed decrease in free ubiquitin and neuronal death. The authors concluded that proteins are severely damaged and aggregated in neurons after focal ischemia. The authors propose that protein damage or aggregation may contribute to ischemic neuronal death.

Animals↗

PubMed2001

Ischemic preconditioning preserves mitochondrial function after global cerebral ischemia in rat hippocampus.

Ischemic tolerance in brain develops when sublethal ischemic insults occur before "lethal" cerebral ischemia. Two windows for the induction of tolerance by ischemic preconditioning (IPC) have been proposed: one that occurs within 1 hour after IPC, and another that occurs 1 or 2 days after IPC. The authors tested the hypotheses that IPC would reduce or prevent ischemia-induced mitochondrial dysfunction. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mm Hg) for 2 and 10 minutes, respectively. Nonsynaptosomal mitochondria were harvested 24 hours after the 10-minute "test" ischemic insult. No significant changes were observed in the oxygen consumption rates and activities for hippocampal mitochondrial complexes I to IV between the IPC and sham groups. Twenty-four hours of reperfusion after 10 minutes of global ischemia (without IPC) promoted significant decreases in the oxygen consumption rates in presence of substrates for complexes I and II compared with the IPC and sham groups. These data suggest that IPC protects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia.

Animals↗

PubMed2001

Human albumin therapy of acute ischemic stroke: marked neuroprotective efficacy at moderate doses and with a broad therapeutic window.

BACKGROUND AND PURPOSE: We examined the neuroprotective efficacy of moderate-dose human albumin therapy in acute focal ischemic stroke and defined the therapeutic window after stroke onset, within which this therapy would confer neurobehavioral and histopathological neuroprotection. METHODS: Sprague-Dawley rats were anesthetized with halothane/nitrous oxide and received 2-hour middle cerebral artery occlusion (MCAo) by a poly-L-lysine-coated intraluminal suture. Neurological status was evaluated during occlusion (60 minutes) and daily for 3 days after MCAo. In the dose-response study, human albumin doses of either of 0.63 or 1.25 g/kg or saline vehicle (5 mL/kg) were given intravenously immediately after suture removal. In the therapeutic window study, a human albumin dose of 1.25 g/kg was administered intravenously at 2 hours, 3 hours, 4 hours, or 5 hours after onset of MCAo. Three days after MCAo, brains were perfusion-fixed, and infarct volumes and brain swelling were determined. RESULTS: Moderate-dose albumin therapy significantly improved the neurological score at 24 hours, 48 hours, and 72 hours and significantly reduced total infarct volume (by 67% and 58%, respectively, at the 1.25- and 0.63-g/kg doses). Cortical and striatal infarct volumes were also significantly reduced by both doses. Brain swelling was virtually eliminated by albumin treatment. Even when albumin therapy (1.25 g/kg) was initiated as late as 4 hours after onset of MCAo, it improved the neurological score and markedly reduced infarct volumes in cortex (by 68%), subcortical regions (by 52%), and total infarct (by 61%). CONCLUSIONS: Moderate-dose albumin therapy markedly improves neurological function and reduces infarction volume and brain swelling, even when treatment is delayed up to 4 hours after onset of ischemia.

Acute Disease↗