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Biomedical subjects

L A Cunningham

Publications and source records attributed to L A Cunningham.

At least 19 recordsLinked to original sources
PubMed2004

Vulnerability of mouse cortical neurons to doxorubicin-induced apoptosis is strain-dependent and is correlated with mRNAs encoding Fas, Fas-Ligand, and metalloproteinases.

Cell surface death receptor-mediated neuronal apoptosis, which is a critical component of neurodegeneration, is modulated by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Doxorubicin (Dox) induces neuronal death by the activation of death receptor pathways. Recently, we demonstrated that Dox-induced neuronal apoptosis is regulated by the balance of MMP-3 and TIMP-3 in rat cortical cultures. Inbred mouse strains exhibit differential susceptibility to cell death stimuli in vivo. Prior to employing transgenic approaches to further investigate the roles of TIMP-3 and MMP-3 in neuronal death, we examined whether inbred mice display strain-dependent vulnerability to Dox. We induced neuronal apoptosis with Dox in primary neuronal cultures established from cerebral cortices of embryonic day 15 C57BL/10 or C57BL/6 mice. Using fluorescence activated cell sorting for neurons, we found that C57BL/6 cortical cultures exhibit a 28% greater neuronal death following Dox treatment than C57BL/10. Real-time PCR of unstimulated cultures revealed that C57BL/10 cortical cultures have reduced basal mRNA levels encoding the pro-apoptotic proteins: Fas, FasL, and TIMP-3, but increased levels of the anti-apoptotic molecule MMP-3 as compared to C57BL/6. Furthermore, C57BL/10 cultures treated with Dox displayed an enhanced induction of mRNA transcripts that encode anti-apoptotic MMPs. These results show that C57BL/10 cortical cultures are more resistant to death receptor-mediated apoptotic cell death as compared to C57BL/6, and suggest that this difference is related to Fas, FasL, and MMP expression. Strain-dependent differences in response to apoptotic stimuli may be an important consideration for developing transgenic models of neurodegeneration.

Animals↗
PubMed2003

Tissue inhibitor of metalloproteinases-3 and matrix metalloproteinase-3 regulate neuronal sensitivity to doxorubicin-induced apoptosis.

Metalloproteinase activity at the cell surface influences cellular sensitivity to extrinsic death vs. survival signals in a variety of cell types, through proteolytic shedding of cell surface signalling molecules. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a unique natural metalloproteinase inhibitor that plays a pro-apoptotic role through its ability to inhibit metalloproteinases that proteolytically cleave death receptors and their ligands from the cell surface. To study the convergence of metalloproteinase activity and death receptor signalling in neurons, we established an in vitro model of neuronal apoptosis utilizing the chemotherapeutic drug, doxorubicin (Dox). Primary cultures established from embryonic rat cerebral cortices displayed robust and selective neuronal apoptosis in response to Dox, an effect that was dependent on the activation of the death receptor, Fas. We demonstrate that both TIMP-3 and matrix metalloproteinase-3 (MMP-3) are constitutively expressed by primary cortical neurons in culture, and selectively modulated Fas-mediated neuronal apoptosis induced by Dox. Metalloproteinase inhibition by TIMP-3 was found to be necessary for Dox-induced neuronal death, whereas addition of active MMP-3 markedly attenuated apoptosis and diminished Fas-Fas ligand interaction at the cell surface. These observations implicate a physiological role for the balance of TIMP-3 and MMP-3 activity at the neuronal surface in regulating death receptor sensitivity. The convergence of metalloproteinase activity and death receptor signalling at the cell surface may influence neuronal cell death vs. survival decisions.

Animals↗
PubMed2001

Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures.

Reperfusion damages the blood-brain barrier (BBB). Matrix metalloproteinases (MMPs) are associated with the opening of the BBB, but their cellular localization and activation mechanisms are uncertain. We used immunohistochemistry to determine the cellular localization of the MMPs in reperfused rat brain, and cell cultures to study their activation. Spontaneously hypertensive rats (SHR) had a 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion for times from 3 h to 21 days. Frozen sections were immunostained with antibodies to gelatinase A (MMP-2), stromelysin-1 (MMP-3), and gelatinase B (MMP-9). Sham-operated control rats showed MMP-2 immunostaining in astrocytic processes next to blood vessels. After 3 h of the onset of reperfusion MMP-2 immunostaining increased in astrocytes. At 24 h immunoreactivity for MMP-3 and MMP-9 appeared. MMP-3 co-localized with activated microglia (Ox-42+) and ischemic neurons (NeuN+). MMP-9 immunostaining was seen at 48 h in endothelial cells, neutrophils, and neurons. At 5 and 21 days intense MMP-2 staining was seen in reactive astrocytes around the ischemic core. Studies of activation of the MMP were done in lipopolysaccharide (LPS)-stimulated astrocyte and microglia cultures. Stimulated astrocytes produced an activated form of MMP-2. When microglia were stimulated, they activated MMP-9. Immunostaining showed MMP-3 in cultures of enriched microglial cells. The hydroxymate-type, MMP inhibitor, BB-1101, blocked the activation of MMP-2 and MMP-9 by LPS in mixed glial cultures. We propose that MMP-2 is normally present in astrocytic end feet, and that during ischemia MMP-9 and MMP-3 are produced. MMP-3 in microglia/macrophages may be activating proMMP-9. Our results show that a differential expression of MMPs by astrocytes, microglia, and endothelial cells at the blood vessels is involved in the proteolytic disruption of the BBB.

Animals↗
PubMed2000

Coupling of AMPA receptors with the Na(+)/Ca(2+) exchanger in cultured rat astrocytes.

Astrocytes exhibit three transmembrane Ca(2+) influx pathways: voltage-gated Ca(2+) channels (VGCCs), the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na(+)/Ca(2+) exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca(2+) concentration ([Ca(2+)](i)); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca(2+)](i) is not known. We demonstrate here that 100 microM AMPA in the presence of 100 microM cyclothiazide (CTZ) causes an increase in [Ca(2+)](i) in cultured cerebral astrocytes that requires transmembrane Ca(2+) influx. This increase of [Ca(2+)](i) is blocked by 100 microM benzamil or 0.5 microM U-73122, which inhibit reverse-mode operation of the Na(+)/Ca(2+) exchanger by independent mechanisms. This response does not require Ca(2+) influx through VGCCs, nor does it depend upon a significant Ca(2+) influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca(2+) stores, although depletion of these Ca(2+) stores does not decrease the peak [Ca(2+)](i) response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca(2+)](i) to increase through the reverse mode operation of the Na(+)/Ca(2+) exchanger with an associated release of Ca(2+) from intracellular stores. This proposed mechanism requires neither Ca(2+)-permeant AMPARs nor the activation of VGCCs to be effective.

Animals↗
PubMed2000

Glial cell line-derived neurotrophic factor in the rat pituitary gland.

The presence of glial cell line-derived neurotrophic factor (GDNF) is described within specific regions of the adult rat pituitary gland. Immune staining methods revealed a small number of GDNF-immunopositive cells in the anterior lobe, and in areas of the neural lobe, while no immunoreactive endocrine cells were observed in the intermediate lobe. In the neural lobe, immunofluorescence methods were also used to demonstrate that GDNF and glial fibrillary acidic protein (GFAP) are co-localized in the glial cells (pituicytes) of the neural lobe. GDNF was not co-localized with neurofilament (NF) in nerve fibers of the neural lobe, suggesting that it is not present in axonal fibers. Measurements of GDNF content in separated anterior and neurointermediate lobes were also performed, using an enzyme-linked immunoassay (ELISA). Values for GDNF were slightly higher in the neurointermediate lobe than those obtained for the anterior lobe. The presence of GDNF in areas of the pituitary is discussed in the context of its possible function to support and maintain hypothalamic innervation, as well as a potential autocrine factor within endocrine cells.

Animals↗
PubMed1999

Sexual dysfunction associated with the treatment of depression: a placebo-controlled comparison of bupropion sustained release and sertraline treatment.

This study compared the sexual functioning effects as well as the safety and efficacy of bupropion sustained release (bupropion SR) and sertraline. Three hundred sixty-four patients with normal sexual functioning and recurrent major depression were treated with bupropion SR (150-400 mg/day), sertraline (50-200 mg/day), or placebo for 8 weeks in this randomized, double-blind, multicenter study. Patients' depression, sexual functioning, and overall safety were assessed at regular clinic visits. Significantly (P < 0.05) more patients treated with sertraline experienced orgasm dysfunction compared with patients treated with bupropion SR or placebo. Bupropion SR, but not sertraline, was statistically significantly superior to placebo in improving scores on all depression scales by the end of the study. Headache occurred with similar frequency in all groups. Gastrointestinal disturbances occurred more frequently with sertraline; insomnia and agitation occurred more frequently with bupropion SR. Small decreases in mean weight were seen with both active treatments; the placebo group experienced a minor increase in mean weight. Both bupropion SR and sertraline were generally well tolerated, although sertraline was more often associated with sexual dysfunction. Bupropion SR, but not sertraline, was statistically superior to placebo in relieving depression by the end of the study. Bupropion SR may offer advantages over sertraline in treating depressed patients concerned with sexual functioning.

Adult↗
PubMed1998

Use of GFAP-lacZ transgenic mice to determine astrocyte fate in grafts of embryonic ventral midbrain.

Embryonic ventral midbrains from GFAP-lacZ transgenic mice were xenografted into the dopamine-depleted striata of adult rats. This transgenic line harbors a nuclear-targeted bacterial beta-galactosidase (beta-gal) reporter gene under transcriptional control of the human glial fibrillary acidic protein (GFAP) promoter sequence. Five weeks post-transplantation, graft-derived astrocytes and dopaminergic neurons were visualized by dual immunocytochemistry for beta-gal and tyrosine hydroxylase (TH), respectively. This report describes the advantages associated with the use of GFAP-lacZ transgenic mice to study astrocyte fate in embryonic neural grafts.

Animals↗
PubMed1997

Human fetal astrocytes as an ex vivo gene therapy vehicle for delivering biologically active nerve growth factor.

The therapeutic use of neurotrophic factors for neurodegenerative diseases is promising, however, optimal methods for continuous delivery of these substances to the human central nervous system (CNS) remains problematic. One approach would be to graft genetically engineered human cells that continuously secrete high levels of a biologically produced and processed neurotrophic factor. This ex vivo gene therapy approach has worked well in animal models of neurodegenerative diseases using a variety of nonneuronal cell types to deliver the transgene. In our studies, we have been investigating the potential of astrocytes, a cell type normally present in the CNS, as a vehicle for ex vivo gene therapy. Here, we demonstrate that astrocytes in the human fetal cortex can be isolated and efficiently infected with an amphotropic retrovirus harboring mouse beta-nerve growth factor (NGF). These transduced astrocytes express high levels of NGF mRNA and secrete bioactive NGF protein as demonstrated by stimulation of neurite outgrowth from adrenal chromaffin cells. NGF ELISA showed that these astrocytes secrete NGF protein at a rate of 41 ng/day per 10(5) cells after 2 weeks in vitro, whereas NGF is undetectable in medium conditioned by normal astrocytes. These data suggest that human fetal astrocytes can be used for delivering biologically produced neurotrophic factors to the human CNS.

Astrocytes↗
PubMed1997

Astrocytes promote or impair the survival and function of embryonic ventral mesencephalon co-grafts: effects of astrocyte age and expression of recombinant brain-derived neurotrophic factor.

Intrastriatal grafting of dopamine-rich embryonic ventral mesencephalon (VM) is a potential therapeutic treatment for Parkinson's disease. However, it has been suggested that the efficacy of this procedure might be improved by enhancing the survival and/or degree of neurite outgrowth by the grafted VM, since these parameters are currently suboptimal. In the present study, we tested the ability of astrocytes retrovirally transduced to produce recombinant brain-derived neurotrophic factor (BDNF) to enhance the survival and/or function of embryonic VM in the unilateral 6-hydroxydopamine (6-OHDA) lesioned rat, a well-characterized rodent model of Parkinson's disease. In culture, primary astrocytes derived from Postnatal Day 0 (P0) rat striatum and transduced with the BDNF vector increased the survival of Embryonic Day 15 (E15) dopaminergic VM neurons by approximately threefold and reduced the loss of dopaminergic neurons following 6-OHDA treatment by approximately 20%. The cultured astrocytes were then mixed 1:1 with freshly dissociated E15 VM and co-grafted into the dopamine-denervated striatum. Unexpectedly, the control nontransduced astrocytes reduced the survival of dopaminergic neurons by 60% and restricted the pattern of neurite outgrowth by the co-grafted VM, compared to grafts of VM alone at 7 weeks postgrafting. These effects were paralleled by an attenuated rate and degree of behavioral recovery. The detrimental effects of the control astrocytes were partially reversed when the astrocytes were transduced to express BDNF, although dopaminergic neuron survival was still reduced by 30% compared to that within VM-only grafts. To begin to assess whether the detrimental effects of the astrocytes were related to the maturational state of the cultured astrocytes, astrocytes were obtained from E18 striatum and maintained in short-term culture (9 days vs several weeks for P0 cultures) prior to co-grafting with VM. Interestingly, the younger astrocytes did not reduce graft survival and allowed for better graft integration. These results suggest that primary astrocytes maintained in long-term culture are detrimental to embryonic neural grafts, an effect that is not completely overcome by expression of recombinant BDNF, and that astrocyte age may be an important consideration in the use of these cells as CNS gene delivery vehicles.

Animals↗
PubMed1997

The generation and maintenance of schedule-induced polydipsia in normal male rats without weight reduction.

Experiment One demonstrated that two normal male Sprague-Dawley rats (approximately 60 days old) with free access to food and two control rats whose weights were held constant by dietary restriction acquired schedule-induced polydipsia (SIP) in daily 33-35 min sessions of fixed-time 60-s food delivery. Three of the rats showed rapid acquisition of SIP; the fourth acquired SIP more slowly and consumed less per session the other three rats. After a 36-40 day period without sessions, the constant-weight rats showed a 37% decrease in overall consumption due to reduced drinking bout length. The SIP of the free-feeding rats was not affected by the interruption. After 90-100 periodic food delivery sessions, all subjects consumed an average of 11.2-12.2 mL per session compared with 1.8-4.8 mL per session in baseline sessions with massed food presentations. Experiment Two replicated the acquisition phase of Experiment One using two non-weight-reduced rats of the age and size of those typically used in SIP studies (approximately 30 weeks old). Both acquired SIP, although one showed only a small average increase in consumption per session over baseline (2.8 mL/session under periodic food vs. 0.8 mL following massed-food presentations). Before weight reduction, the stronger drinker consumed approximately 8.8 mL per session compared with an average of 0.6 mL per session in baseline. After weight reduction, both exhibited strong SIP (18-19 mL per session in the final five sessions). This study demonstrates that weight reduction is not a necessary condition for the generation and maintenance of SIP in rats.

Animals↗
PubMed1997

Once-daily venlafaxine extended release (XR) and venlafaxine immediate release (IR) in outpatients with major depression. Venlafaxine XR 208 Study Group.

This was a randomized, double-blind, placebo-controlled comparison of the efficacy and safety of once-daily venlafaxine extended release (XR) and venlafaxine immediate release (IR). Outpatients with DSM-III-R major depression were randomly assigned to venlafaxine XR, 75 mg once daily, venlafaxine IR, 37.5 mg twice daily, or placebo for a maximum of 12 weeks. If the response was inadequate after 2 weeks of treatment, the dosage of venlafaxine XR or IR could be increased to 150 mg daily. The primary efficacy variables were the 21-item Hamilton Depression (HAM-D) Rating Scale total score and depressed mood item, the Montgomery-Asberg Rating Scale (MADRS) total scores, and the Clinical Global Impressions (CGI) severity scale. Two hundred seventy-eight patients were evaluated for efficacy. Venlafaxine XR was significantly superior (p < 0.05) to placebo beginning at week 2 for the HAM-D, week 3 for the MADRS, and week 4 for the CGI severity. Similarly, venlafaxine IR was significantly superior (p < 0.05) to placebo beginning at week 2 on the HAM-D total and depressed mood item, week 3 on the MADRS total, and week 6 on the CGI severity scales. Venlafaxine XR exhibited superiority (p < 0.05) over venlafaxine IR at week 12 for all efficacy variables. The most common treatment-emergent adverse event with venlafaxine XR was nausea. The incidence of nausea was highest during the first 2 weeks with a low likelihood of developing nausea thereafter. The results of this study indicate that venlafaxine XR is safe, effective, and well tolerated for the treatment of major depression at once-daily doses ranging from 75 to 150 mg.

Adolescent↗
PubMed1997

Depression & anxiety in the primary care setting.

Depression and anxiety disorders are common in primary care. Diagnosis is accomplished by applying of specific, data-driven diagnostic criteria published in the DSM-IV. The differential diagnosis is sometimes difficult, but as a general rule the treatment of choice for both conditions is antidepressant medicine. It is very important to remember that anxiety disorders respond very nicely to most antidepressants, but depression does not respond to tranquilizers. BZs, should be reserved for adjunctive use in the early stages of treatment for both anxiety disorders and depression. The azapirone, buspirone, may be used as first line treatment for generalized anxiety disorder and may also augment antidepressants when the patient presents with mixed anxiety and depression. The important point is that when in doubt about either the diagnosis or which drug to choose first it is generally safe to select one of the newer antidepressants discussed in this article.

Anti-Anxiety Agents↗
PubMed1996

The oral dose-effect relationship for fluvoxamine: a fixed-dose comparison against placebo in depressed outpatients.

This 7- to 8-week, multicenter, randomized, double-blind, placebo-controlled study was performed to determine the dose-effect relationship and minimum effective dose for fluvoxamine maleate in a titrated fixed-dose study of major depressive disorder. Gradual titration over 2 weeks to fixed maintenance doses was employed to minimize dropout due to initial side effects. The study enrolled 600 outpatients, male and female, age 18-65, meeting DSM-III-R criteria for major depressive disorder. A 13-item subscore of the standard 21-Item Hamilton Depression Scale was used to minimize the possible contribution of known side effects from serotonin reuptake inhibitors to the overall HAM-D score. Secondary efficacy assessments included the HAM-D retardation factor, HAM-D depressed mood item, CGI-severity of illness item, and SCL depression factor. Fluvoxamine (50-150 mg/day) was therapeutically effective and well tolerated during 6 weeks of therapy. Based on the HAM-D depressed mood item, efficacy was dose dependent. The minimum effective dose was 50 mg/day. Fluvoxamine maleate shows dose-related effectiveness in the acute treatment of major depressive disorder.

Administration, Oral↗
PubMed1994

In vitro studies of glucocorticoid effects on neurons and astrocytes.

Studies using immunocytochemistry and RNase protection assay demonstrate that glucocorticoid and mineralocorticoid receptors (GR, MR) and their corresponding mRNAs are co-expressed in hippocampal neurons cultured in serum-free, defined medium and at lower levels in cultured astrocytes. Addition of serum or medium conditioned by astrocytes increases the levels of MR mRNA, but has little effect on the levels of GR mRNA. Cellular levels of both GR mRNA and MR mRNA are upregulated by growth of embryonic hippocampal neurons in corticosterone. This is in distinct contrast to regulation of receptor expression in vivo where mRNAs for these receptors are downregulated in the rat hippocampus by corticosterone treatment of the adult adrenalectomized rat. However, in cultured astrocytes, GR and MR mRNAs are also downregulated by corticosterone. To begin to define the role of glucocorticoids in gene expression in astrocytes, we have used giant two-dimensional (2D) gel electrophoresis to separate astrocyte cellular proteins and translation products synthesized in vitro from astrocyte poly A+ RNA. Analysis of approximately 1,500 in vitro translation products by giant 2D gel electrophoresis reveals 11 protein inductions and 1 repression that occur at the level of mRNA in the absence of protein synthesis following treatment of astrocytes with corticosterone. Interestingly, these changes appear to be mediated by GR, but not by MR. The in vitro studies described here are relevant to identifying the role of GR and MR in gene expression in specific cell types in the hippocampus.

Animals↗
PubMed1994

Nerve growth factor released by transgenic astrocytes enhances the function of adrenal chromaffin cell grafts in a rat model of Parkinson's disease.

Previous studies have demonstrated that astrocytes genetically modified to express recombinant nerve growth factor (NGF) support the survival and neuronal transdifferentiation of intrastriatal adrenal chromaffin cell grafts at 2 weeks post-transplantation [15]. The present study was performed to determine whether these effects would be maintained at longer times post-transplantation and, if so, whether the co-grafts would reduce rotational behavior in the unilateral 6-hydroxydopamine-lesioned rat. In the present study, we have demonstrated that primary type I rat astrocytes infected with a replication-defective retrovirus conferring expression of a mouse beta-NGF cDNA sequence secrete NGF at a rate that is approximately 40-fold higher than that of controls (i.e., 8.0 vs. 0.2 pg NGF/h/10(5) cells, respectively). The genetically modified astrocytes were also found to express recombinant NGF following intrastriatal transplantation, as indicated by a 23% increase in striatal NGF content compared with controls, measured at 4 weeks post-transplantation. When NGF-producing astrocytes and adrenal chromaffin cells were co-grafted into the dopamine-denervated striatum of the unilateral 6-hydroxydopamine-lesioned rat, the chromaffin cells displayed extensive neurite outgrowth and a 5-12-fold increase in survival compared to controls at 10 weeks post-grafting. These effects were paralleled by a 60% reduction of apomorphine-induced rotational behavior, suggesting a partial normalization of striatal function. These results suggest that genetically modified astrocytes promote the prolonged survival and function of adrenal chromaffin cell grafts in a rat model of Parkinson's disease.

Adrenal Medulla↗
PubMed1994

Depression in the medically ill: choosing an antidepressant.

Choosing an antidepressant for a medically ill patient is a common clinical problem. There is an increased prevalence of depression among patients with many medical illnesses. The clinician must be familiar with the recent additions to the antidepressant armamentarium, including the synaptic pharmacology and pharmacokinetics of the available agents. The factors usually used to guide antidepressant selection must be augmented by a careful evaluation of the patient's medical illness and therapeutic regimen. Special attention must be paid to the potential for drug-illness and drug-drug interactions. The author has used the example of four illnesses commonly associated with depression--stroke, cancer, Parkinson's disease, and dementia--to illustrate how these considerations affect the choice of an antidepressant.

Antidepressive Agents↗
PubMed1994

A comparison of venlafaxine, trazodone, and placebo in major depression.

A double-blind, placebo-controlled trial was undertaken to compare the safety and efficacy of venlafaxine and trazodone in patients with major depression. Two hundred twenty-five patients entered an initial 6-week treatment phase, and 149 completed it. Ninety-six patients who were responders continued in a 1-year, double-blind, long-term phase during which they received the same medication and doses they had during the short-term phase. Both active treatments were significantly more effective than placebo on some measures during the short-term study, but venlafaxine produced more improvement in the cognitive disturbance and retardation factors on the Hamilton Rating Scale for Depression. Trazodone was more effective against the sleep disturbance factor. Patients on venlafaxine were most likely to enter the long-term phase and to remain in the trial longest. The side effect profiles of the three treatment groups were compared. Venlafaxine was most likely to cause nausea, whereas trazodone was associated with the most dizziness and somnolence.

Adult↗