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J W Simpkins

Publications and source records attributed to J W Simpkins.

At least 37 records · Page 2Linked to original sources
PubMed1999

Pre-exposure of cells to cationic lipids enhances transgene delivery and expression in a tissue culture cell line.

Several factors influence non-viral transfection in tissue culture models including nature of the cationic lipid, plasmid construction, and DNA lipid complex, among others. The cell line itself is another confounding variable. Each subcellular population may respond independently to the transgene or specific delivery vector with regards to toxicity or transgene expression. In this study, the SKnSH (human neuroblastoma) and COS-1 (African green kidney) cells were exposed to three different treatments A, B, and C. Treatment A refers to cells obtained from American Type Culture Collection (ATCC) and cultivated as recommended, treatment B to cells that were grown in presence of cationic lipids for two weeks, and treatment C to cells that were grown in presence of cationic lipids for two weeks followed by normal media for two weeks to determine if lipid mediated effects were reversible. Treatment B resulted in a three-fold increase in transgene expression of a reporter gene as compared to the other treatments. This increase in transgene expression appeared not to be related to alterations in toxicity. Interestingly, the fluid phase endocytic uptake of fluorescently labeled oligonucleotides was increased in treatment B. However, there was no significant difference in the cellular-associated signal when fluorescently labeled plasmid-DNA was evaluated. In COS-1 cells, no difference in transfection was observed with treatment B illustrating that cell lines respond independently. In conclusion, pre-exposure of SKnSH cells to cationic liposomes (treatment B) resulted in higher transgene production.

Animals↗
PubMed1998

Estrogen attenuates over-expression of beta-amyloid precursor protein messager RNA in an animal model of focal ischemia.

Cerebral ischemia is a risk factor for late onset Alzheimer's disease. Since estrogen replacement therapy benefits the outcome of cerebral stroke in post-menopausal women, we designed the present study to investigate the effects of estrogen on the expression of beta-amyloid precursor protein (APP) mRNA following focal ischemia in female rats. Female rats were ovariectomized (OVX) for two weeks. A single dose of 17 beta-estradiol (E2) (100 microgram/kg) was injected s.c. two hours before a unilateral middle cerebral artery (MCA) occlusion. Brain samples were harvested from ischemic core and penumbra of cortices at one hour and twenty-four hours following MCA occlusion. The expression of APP mRNA was assessed by RT-PCR. At one hour after MCA occlusion, OVX rats had a 67.9% (p<0.05) increase in APP mRNA in the penumbra. E2 treatment reduced this APP mRNA over-expression by 26.3% at that region. At twenty four hours following MCA occlusion, OVX rats had increases in APP mRNA of 52.9% and 57.0% (p<0.05) in the core and penumbra, respectively. E2 treatment reduced the APP mRNA over-expression by 61.0% and 48.6% (p<0.05) in these two regions, respectively. These effects appeared to reflect an interaction between hormonal environment and ischemia, since in the absence of MCA occlusion, there were no significant differences in APP mRNA expression among OVX, OVX-E2 treated and intact female rats. The present study demonstrates that estrogen may have an important role in reducing the over-expression of APP mRNA following focal ischemia.

Amyloid beta-Protein Precursor↗
PubMed1998

Testosterone increases and estradiol decreases middle cerebral artery occlusion lesion size in male rats.

This study was undertaken to determine the effects of estrogen and testosterone on cerebral ischemic lesion size induced by middle cerebral artery (MCA) occlusion in male rats. Rats were gonadectomized and treated with testosterone, estrogen, or testosterone plus estrogen filled Silastic pellets. The animals were divided into 6 groups: intact, intact + estrogen (E2), castrate, castrate + testosterone (T), castrate + E2, and castrate + T + E2. One week after treatment, cerebral ischemia was induced by MCA occlusion for 40 min, followed by reperfusion. After 24 h, rats were sacrificed and slices were then stained to assess lesion size. The presence of testosterone increased and the removal of testosterone decreased lesion size. A strong positive correlation (r2 = 0.922) between plasma testosterone concentrations and ischemic lesion size was observed. Estradiol treatment reduced ischemic area. In summary, the present study provides evidence that testosterone exacerbates and estrogens ameliorate ischemic brain damage in an animal model of cerebral ischemia.

Animals↗
PubMed1998

Effects of gender and estradiol treatment on focal brain ischemia.

The present studies were undertaken to investigate the effects of gender and estrogen treatment on focal cerebral ischemia in male and female rats. Focal ischemia was created by inserting a 3-0 surgical suture through the left cervical internal carotid artery to obstruct the blood flow into the middle cerebral artery (MCA). The MCA was reperfused by removing the suture in 40 min. All rats were sacrificed for measurement of infarct area after 24 h. In the first study, mortalities from MAC occlusion were 12.5% (2/16) each for intact male rats and intact female rats, and 23.5% (4/17) for ovariectomized (OVX) female rats. The coronal infarct area (mean+/-S. E.M.) was 9.5+/-1.0% for intact female rats, 16.6+/-1.6% for intact male rats (p=0.0001 vs. intact female rats), and 16.0+/-1.4% for OVX female rats (p=0.0002 vs. intact female rats). In a second experiment, OVX-female rats were administrated either 17beta-estradiol (E2) or its vehicle, hydroxypropyl-beta-cyclodextrin (HPCD), at 40 min after the onset of MCA occlusion. Mortalities were 40% (4/10) for vehicle treated OVX rats and 0% for E2 treated OVX rats. The coronal infarct area (mean+/-S.E.M.) was 19.3+/-1.8% for vehicle treated rats vs. 8.0+/-1. 2% for E2 treated rats (p<0.01). Serum estrogen levels for vehicle treated OVX rats were 14.5+/-1.2% pg/ml vs. 142.7+/-23.6 pg/ml for E2 treated OVX rats (p<0.01). These results strongly suggest that the level of circulating estrogens play an important role in protecting brain tissues against ischemia induced by MCA occlusion.

Animals↗
PubMed1998

Hypoglycemia-induced seizures reduce cyclic AMP response element binding protein levels in the rat hippocampus.

Cyclic AMP response element binding protein (CREB) is a transcription factor that has been implicated in the activation of protein synthesis required for long-term memory. Since memory deficits are manifest following seizure, we undertook the present study to investigate the effects of hypoglycemia-induced seizure on CREB-immunoreactive neurons in several brain regions. We induced generalized seizures in male Long Evans rats (n=5) by injecting them with insulin (30 IU/kg, i.p). Animals were recovered by administration of 3 ml of 30% glucose within 5 min of the occurrence of seizure. Control animals (n=3) were injected with saline instead of insulin. All animals were perfused 90 min after recovery and the brains processed for CREB immunohistochemistry. Cell counts were determined for CREB-positive neurons using a computer-assisted program. When compared to control animals there was a 50% decrease (P<0.0001) in CREB-positive neurons in the CA1 region of the experimental animals. In the CA3 and dentate gyrus there was a 36% (P<0.001) and 25% decrease (P<0.001), respectively. Given the importance of hippocampus in memory-related processes and evidence that CREB is critical for memory formation, it is possible that seizures interfere with memory by disrupting CREB-dependent transcription.

Animals↗
PubMed1998

Nuclear estrogen receptor-independent neuroprotection by estratrienes: a novel interaction with glutathione.

Post-menopausal estrogen replacement therapy is associated with a reduction in the risk of Alzheimer's disease and has been reported to improve cognitive functioning in several small clinical trials. The present study evaluates the dependence of estrogenic neuroprotection on the presence of estrogen receptors using the murine neuronal cell line, HT-22, exposed to the neurotoxic beta-amyloid peptide. These cells lack functional estrogen receptors. The amyloid peptide killed 50-60% of these cells and concurrent treatment with either of three estratrienes, beta-estradiol, alpha-estradiol, or estratrien-3-ol, resulted in a dose-dependent protection. The potency of this estrogen neuroprotection was dependent on the presence of glutathione in the culture media. The presence of reduced glutathione in the media increases the neuroprotective potency of estrogens by an average of 400-fold. These results demonstrate that a nuclear estrogen receptor is not necessary for the neuroprotective actions of estrogens; however, the presence of an appropriate antioxidant in the extracellular milieu is needed for estratriene neuroprotection at physiologically and pharmacologically relevant doses. These data suggest the possibility of combined estrogen-antioxidant therapy for neurodegenerative diseases such as Alzheimer's disease.

Amyloid beta-Peptides↗
PubMed1998

A novel, synergistic interaction between 17 beta-estradiol and glutathione in the protection of neurons against beta-amyloid 25-35-induced toxicity in vitro.

The present studies were undertaken to investigate the possibility of an interaction between 17 beta-estradiol (E2) and glutathione in protecting cells against the presence of beta-amyloid 25-35 (betaAP 25-35). We demonstrate that when evaluated individually, supraphysiological concentrations of either E2 (200 nM) or of reduced glutathione (GSH; 325 microM) can protect SK-N-SH human neuroblastoma cells from betaAP 25-35 (20 microM) toxicity. This dose of betaAP 25-35 was chosen based on the LD50 (28.9 microM) obtained in our earlier work. However, in the presence of 3.25 microM GSH, the neuroprotective EC50 of E2 was shifted from 126 +/- 89 nM to 0.033 +/- 0.031 nM, approximately 4000-fold. Similarly, in primary rat cortical neurons, the addition of GSH (3.25 microM) increased the potency of E2 against betaAP 25-35 (10 microM) toxicity, as evidenced by a shift in the EC50 values of E2 from 68 +/- 79 nM in the absence of GSH to 4 +/- 6 nM in its presence. The synergy between E2 and GSH was not antagonized by the addition of the estrogen receptor antagonist, ICI 182,780. Other thiol-containing compounds did not interact synergistically with E2, nor were any synergistic interactions observed between E2 and ascorbic acid or alpha-tocopherol. Based on these data, we propose an estrogen-receptor independent synergistic interaction between glutathione and E2 that dramatically increases the neuroprotective potency of the steroid and may provide insight for the development of new treatment strategies for neurodegenerative diseases.

Amyloid beta-Peptides↗
PubMed1997

Low concentrations of estradiol reduce beta-amyloid (25-35)-induced toxicity, lipid peroxidation and glucose utilization in human SK-N-SH neuroblastoma cells.

The present studies were undertaken to determine the role of physiologically relevant concentrations of estrogens on amyloid-induced changes in cell viability, metabolic demands, and lipid peroxidation in response to the toxic fragment of beta-amyloid (betaAP 25-35). To this end, SK-N-SH human neuroblastoma cells were exposed to betaAP 25-35 or betaAP 25-35 plus 17beta-estradiol, and cell viability, media glucose use and lactate production were measured at time points ranging from 3 to 15 h for examination of acute effects, or at 48 and 72 h time points for chronic effects. Addition of betaAP 25-35 to SK-N-SH cells decreased the number of viable cells from 5% at 3 h to 35% at 15 h when compared to vehicle controls. Chronic treatment for 48 and 72 h caused decreases in viable cell number of 70% and 65%, respectively. Paradoxically, both glucose utilization and lactate production were found to be increased for the betaAP-treated cells. Concomitant estrogen treatment was found to be neuroprotective, as the severity of the insult on cell viability was decreased by 40% at 15 h and up to 71% at 72 h. Likewise, the addition of 17beta-estradiol decreased both the glucose use and lactate production of the cells. Chronic treatment with betaAP caused increases in lipid peroxidation over vehicle treated controls of 82% and 78% at 48 and 72 h, respectively, while decreases in peroxidation of 48% were seen with simultaneous estrogen treatment. These results indicate that the neuroprotective effects of estrogens against betaAP-induced toxicity are due in part to their capability to decrease lipid peroxidation and may additionally be attributable to decreasing the metabolic load of the cell.

Amyloid beta-Peptides↗
PubMed1997

Hypoglycemia enhances the expression of mRNA encoding beta-amyloid precursor protein in rat primary cortical astroglial cells.

Deposition of beta-amyloid (A beta) is a characteristic feature of the pathology of Alzheimer's disease (AD). Since glucose metabolism and the consequential ATP production are depressed in the temporal and parietal regions of the cortex in patients with AD, we designed the present study to investigate the possible role of hypometabolism in the pathogenesis of AD. We incubated rat primary cortical astroglial cells for 2 h to 4 days in a media deprived of 95% of its glucose and assessed the expression and alternative splicing of the mRNA that encoding beta-amyloid precursor protein (APP) using RT-PCR. Hypoglycemia caused a time-dependent increase in APP mRNA expression, which reaches a peak level of 173.2% of control expression (P < 0.05) at 24 h of hypoglycemia. Noteworthy, hypoglycemia favors the alternative splicing that includes the exon 7 segment, which encodes a Kunitz-type serine protease inhibitor domain. This study demonstrates that hypoglycemia increases APP mRNA expression in astroglial cells and processing of APP mRNA to a form that may encourage A beta deposits in AD. These data suggest that the observed hypometabolism in AD may contribute to its deposition of A beta in affected brain regions.

Amyloid beta-Protein Precursor↗
PubMed1997

Role of estrogen replacement therapy in memory enhancement and the prevention of neuronal loss associated with Alzheimer's disease.

Recent evidence supports a role for estrogens in both normal neural development and neuronal maintenance throughout life. Women spend 25-33% of their life in an estrogen-deprived state and retrospective studies have shown an inverse correlation between dose and duration of estrogen replacement therapy (ERT) and incidence of Alzheimer's disease (AD), suggesting a role for estrogen in the prevention and/or treatment of neurodegenerative diseases. To explore these observations further, an animal model was developed using ovariectomy (OVX) and ovariectomy with estradiol replacement (E2) in female Sprague-Dawley rats to mimic postmenopausal changes. Using an active-avoidance paradigm and a spatial memory task, the effects of estrogen deprivation were tested on memory-related behaviors. OVX caused a decline in avoidance behavior, and estrogen replacement normalized the response. In the Morris water task of spatial memory, OVX animals showed normal spatial learning but were deficient in spatial memory, an effect that was prevented by estrogen treatment. Together these data indicate that OVX in rats results in an estrogen-reversible impairment of learning/memory behavior. Because a plethora of information has been generated that links decline in memory-related behavior to dysfunction of cholinergic neurons, the effects of estrogens on cholinergic neurons were tested. We demonstrated that OVX causes a decrease in high affinity choline uptake and choline acetyltransferase activity in the hippocampus and frontal cortex; ERT reverses this effect. Further, we showed that estrogens promote the expression of mRNA for brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), 2 neurotrophic substances that have been shown to ameliorate the effects of age and injury on cholinergic neurons. Tissue culture models were used to evaluate whether estrogen treatment increases the survival of neurons when exposed to a variety of insults. 17-beta-Estradiol (beta-E2) protects cells from the neurotoxic effects of serum deprivation and hypoglycemia in human neuroblastoma cell lines. We have also observed that 17-alpha-estradiol (alpha-E2), a weak estrogen, shows neuroprotective efficacy in the SK-N-SH cell line at concentrations equivalent to beta-E2. Finally, we have observed that tamoxifen, a classic estrogen antagonist, blocks only one-third of the neuroprotective effects of either alpha-E2 or beta-E2. Collectively, these results indicate that estrogen is behaviorally active in tests of learning/ memory; activates basal forebrain cholinergic neurons and neurotrophin expression; and is neuroprotective for human neuronal cultures. We conclude that estrogen may be a useful therapy for AD and other neurodegenerative diseases.

Alzheimer Disease↗
PubMed1997

17 alpha-estradiol exerts neuroprotective effects on SK-N-SH cells.

Estradiol (E2) has been shown to exert organizational, neurotrophic, and neuroprotective effects in the CNS. The present study assessed the specificity of the neuroprotective effects of estradiol for the potent 17 beta-isomer. SK-N-SH cells from a human neuroblastoma cell line, which we have shown to be estrogen-responsive, were cultured at low or high plating density. Then cells were exposed to 17 beta-E2 (0.2 or 2 nM), 17 alpha-E2 (0.2 or 2 nM), or cholesterol, testosterone, dihydrotestosterone, progesterone, or corticosterone (all at 2 nM). Cultures were insulted by serum deprivation, which caused a profound loss of cells. At 1 or 2 d of serum deprivation and steroid hormone replacement, the protection afforded cells by the steroid addition was assessed. Serum deprivation killed approximately 90% of cells cultured at both low and high plating density. Both 17 alpha- and 17 beta-E2 provided protection of SK-N-SH cells at either plating density. Further, a 10-fold molar excess of tamoxifen antagonized only approximately one-third of the neuroprotective effects of either isomer of estradiol, and a 100-fold excess of tamoxifen had no additional effect on the neuroprotection by 17 beta-E2. By contrast, none of the other steroids tested protected cells from the insult of serum deprivation. These results indicate that the neuroprotective effects of estrogens are not attributable to the general steroid structure, and the majority of the neuroprotection may not be mediated via a tamoxifenantagonized receptor mechanism.

Cholesterol↗
PubMed1997

17 beta-estradiol attenuates fimbrial lesion-induced decline of ChAT-immunoreactive neurons in the rat medial septum.

We investigated the neuroprotective effects of 17 beta-estradiol (E2) on medial septal cholinergic neurons following partial unilateral lesion of the fimbriafornix. Adult female rats were ovariectomized (OVX) and, 5 days later, treated with a single intravenous (iv) injection of an estradiol (E2)-chemical delivery system (E2-CDS) or its vehicle hydroxypropyl-beta-cyclodextrin (HPCD). All rats were subjected to partial unilateral electrolytic fimbrial lesion the following day. At 20 days postlesion, brain slices from treated animals were assessed for choline acetyltransferase (ChAT) by immunohistochemistry. Animals treated with HPCD or E2-CDS showed a 44 or 4% decrease, respectively, in ChAT-positive neurons on the lesioned side compared to the nonlesioned side of the medial septum. In a second study using the same lesioning procedure, adult OVX rats received either a subcutaneous E2 pellet implant (n = 6), or, 5 days postovariectomy, a single iv injection of E2-CDS (n = 8) or HPCD (n = 6). Animals treated with HPCD showed a 55% decrease in ChAT-positive neurons on the lesioned side compared to the nonlesioned side of the medial septum. By contrast, rats treated with E2-CDS or E2 pellet had a 14 or 13% decrease, respectively, in ChAT-positive neurons. Interestingly, E2 treatment substantially decreased ChAT-positive neurons on the nonlesioned side of the medial septum in comparison to control animals. The present study suggests that cholinergic neurons in the medial septum are protected from lesion-induced degeneration by treatments which increase brain E2 levels. Thus, E2 may play a neuroprotective role in the basal forebrain cholinergic system.

Animals↗
PubMed1997

Effects of 17beta-estradiol on glucose transporter 1 expression and endothelial cell survival following focal ischemia in the rats.

Estrogen replacement therapy in postmenopausal women is associated with a decreased mortality and morbidity from stroke. The present study was undertaken to investigate the effects of estrogen on endothelial cell glucose transporter 1 (GLUT 1) and on the cell viability during focal ischemia in a rat model. Female rats were ovariectomized (OVX) and 2 weeks later 17beta-estradiol (E2) was injected subcutaneously at a dose of 100 microg/kg 2 h before unilateral middle cerebral artery (MCA) occlusion. Ischemic lesion size was quantified using 2,3,5-triphenyl tetrazolium chloride (TTC) staining and GLUT 1 protein was analyzed by Western blotting. E2 treatment decreased ischemic lesion size in slices taken at 9 and 11 mm posterior from the olfactory bulb by 46.3% and 44.1%, respectively (P < 0.05). GLUT 1 protein decreased in both OVX and E2 groups by 24.6% and 22.7% respectively (P < 0.05) compared with the non-lesioned side in the core ischemic region, including the basal ganglia. GLUT 1 protein was increased in the E2-treated group compared with the control group (23.3%, P < 0.05) in the penumbral ischemic region of the cortex. Primary rat brain capillary endothelial cell (BCEC) cultures were established as an in vitro model for ischemic effects on endothelial cells. Estrogen reduced BCEC loss by 35.9%, 28.4% and 23.5% (P < 0.05) when glucose in the culture medium was reduced to 50%, 20% and 10%, respectively; and by 28.4% and 18.4% (P < 0.05) following 1 or 4 h of anoxia, respectively. This study demonstrates that estrogen treatment increases GLUT 1 transporters and protects BCEC loss which may in turn reduce focal ischemic brain damage.

Animals↗
PubMed1997

Luteinizing hormone-releasing hormone (LHRH) attenuates morphine-induced inhibition of cyclic AMP (cAMP) in opioid-responsive SK-N-SH cells.

SK-N-SH cells were used to assess the effects of luteinizing hormone-releasing hormone (LHRH) on opioid receptor-mediated changes in cyclic AMP (cAMP). Prostaglandin E1 (PGE1, 1 microM) caused a dramatic increase in cAMP levels. Treatment with 10 microM morphine (MOR) significantly inhibited the stimulatory effect of PGE1, LHRH (0.8 microM) caused an increase in the basal level of intracellular cAMP and potentiated the stimulatory effect of PGE1 on cAMP accumulation. In cells pretreated with LHRH the inhibitory effect of MOR on cAMP accumulation was significantly attenuated. An LHRH antagonist had no effect on cAMP. The involvement of pertussis toxin (PTX)-sensitive G proteins in the actions of LHRH was studied. PTX increased the stimulatory effect of PGE1 on cAMP and attenuated the inhibitory effect of MOR. However, PTX pretreatment prevented the effects of LHRH on the intracellular actions of PGE1 but exerted an additive effect with LHRH in blocking the MOR-induced decrease in cAMP levels. We conclude that LHRH attenuates the inhibitory, opioid receptor-mediated effect of MOR on intracellular cAMP accumulation in SK-N-SH cells, and that the G protein-independent mechanism may be involved in LHRH-induced attenuation of the inhibitory effect of MOR on neuronal cAMP.

Alprostadil↗
PubMed1997

17 beta-Estradiol modulation of glucose transporter 1 expression in blood-brain barrier.

The present study was designed to evaluate 17 beta-estradiol (E2) modulation of glucose transporter 1 (GLUT-1) protein and mRNA expression in blood-brain barrier (BBB) endothelium. Female rats were ovariectomized (OVX) for 12-14 days, then E2 was injected at dosages of 1-100 micrograms/kg sc at 2-16 h before sampling. Glucose transport into BBB endothelial cells was assessed using 2-deoxy-[14C]glucose (2-[14C]DG) uptake. GLUT-1 protein and mRNA samples were analyzed by Western and Northern blotting, respectively. E2 treatment caused dose- and time-dependent increases in 2-[14C]DG uptake and GLUT-1 protein expression by microvessels. The peak responses were induced by 10 micrograms/kg E2 dose at the 4-h sampling time (36.0 and 31.3% increases, P < 0.05, respectively). GLUT-1 mRNA demonstrated a transient increase at 15 min (55%, P < 0.05), then decreased to basal level by 2 h. This study shows that in vivo treatment with E2 increases 2-[14C]DG uptake into the BBB endothelial cells and suggests this E2 effect is due to its modulation of GLUT-1 mRNA and protein.

Animals↗
PubMed1997

Estrogens may reduce mortality and ischemic damage caused by middle cerebral artery occlusion in the female rat.

The present study was undertaken to determine if estrogens protect female rats from the neurodegenerative effects of middle cerebral artery (MCA) occlusion. The rats were ovariectomized and 7 or 8 days later various estrogen preparations were administered before or after MCA occlusion. Pretreatment with 17beta-estradiol (17beta-E2) or a brain-targeted 17beta-E2 chemical delivery system (CDS) decreased mortality from 65% in ovariectomized rats to 22% in 17beta-E2-treated and 16% in 17beta-E2 CDS-treated rats. This marked reduction in mortality was accompanied by a reduction in the ischemic area of the brain from 25.6+/-5.7% in the ovariectomized rats to 9.8+/-4% and 9.1+/-4.2% in the 17beta-E2-implanted and the 17beta-E2 CDS-treated rats, respectively. Similarly, pretreatment with the presumed inactive estrogen, 17alpha-estradiol, reduced mortality from 36 to 0% and reduced the ischemic area by 55 to 81%. When administered 40 or 90 minutes after MCA occlusion, 17beta-E2 CDS reduced the area of ischemia by 45 to 90% or 31%, respectively. In summary, the present study provides the first evidence that estrogens exert neuroprotective effects in an animal model of ischemia and suggests that estrogens may be a useful therapy to protect neurons against the neurodegenerative effects of stroke.

Animals↗
PubMed1997

17beta-estradiol attenuates CREB decline in the rat hippocampus following seizure.

Cyclic AMP response element-binding protein (CREB) is a transcription factor that has been implicated in the activation of a number of genes. We reported that CREB levels decline following a severe hypoglycemic episode in the hippocampus and cortex in the male rat brain. The present experiment was undertaken to investigate whether 17beta-estradiol prevents the decline in CREB-immunoreactive cells following seizure in female rats. Rats were divided into four groups: ovariectomized (OVX), ovariectomized and insulin-treated (OVX-I), estrogen-replaced (E2), and estrogen-replaced and insulin-treated (E2-I). Generalized seizures were induced by injections with insulin (12.5 IU/kg, intraperitoneally) and animals were recovered by administration of glucose within 5 min of the occurrence of seizure. Control animals were injected with saline instead of insulin. All animals were perfused 90 min after recovery and the brains were processed for CREB immunoreactivity. CREB-positive neurons were counted using a computer-assisted program. Insulin treatment of OVX rats caused a significant decline in CREB-positive neurons in the CA1, CA3, and dentate gyrus compared to OVX rats. Estrogen treatment of OVX rats significantly increased CREB-positive neurons in the CA1 and dentate gyrus and attenuated the insulin-induced decline of CREB-positive neurons in all three regions compared to OVX rats. In conclusion, estrogens appear to induce CREB expression and attenuate its decline in the hippocampus following a severe hypoglycemic episode.

Animals↗