Gopal Rajakumar

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 Alzheimers 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.

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.

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.

17 β-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.

17β-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.

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.

Desipramine desensitizes β-adrenergic signal transduction in salivary glands : differential regulation with age

We previously reported that the tricyclic antidepressant, desipramine desensitizes beta-adrenergic signal transduction in parotid and submandibular salivary glands. To determine the consequences of repeated desipramine administration on beta-adrenergic signal transduction in salivary glands from aged rats and whether the recovery after drug withdrawal is impaired, we assessed the effects of 28-day desipramine administration and the reversibility of this treatment following a 15-day washout period on beta-adrenoceptors and adenylyl cyclase activity in parotid and submandibular glands from F-344 rats of 6, 12 and 24 months of age. beta-Adrenoceptors were also assessed in the cerebral cortex. Desipramine administration down-regulated receptor number and attenuated isoproterenol-stimulated adenylyl cyclase activity in all three ages of rats. However, the reduction in isoproterenol-stimulated adenylyl cyclase activity was greater than the loss of receptor number. Desipramine administration attenuated the efficacy of NaF-stimulated activity with no change in forskolin-stimulated adenylyl cyclase activity. These data suggest that in addition to desensitizing beta-adrenergic-mediated signal transduction, desipramine impaired G-protein-mediated adenylyl cyclase stimulation. The recovery from desipramine desensitization was age dependent. beta-Adrenoceptor density recovered more slowly in the cerebral cortex and the submandibular gland in 24-month-old rats than in 6-month-old rats. In contrast, in 12-month-old rats, there was a receptor up-regulation and adenylyl cyclase supersensitivity. These data indicate that the capacity for receptor modulation is age dependent and suggest that desipramine treatment may down-regulate stimulatory G protein.

Desipramine desensitizes β-adrenergic signal transduction in rat salivary glands

Abstract The consequences of the tricyclic antidepressant drugs include sedation, orthostatic hypotension and salivary dysfunction. It was reported that administration of desipramine resulted in a decrease in the concentration of secreted salivary protein. Tricyclic antidepressants may indirectly alter salivary function as a result of their action on the central nervous system to decrease adrenergic neural transmission or, alternately, may act directly on salivary glands to modulate β-adrenergic signal transduction. To investigate the latter possibility, the effects of administration of desipramine (DMI) for 28 days was assessed on β-adrenergic and post-receptor signal transduction in the parotid and submandibular glands of the rat and the reversibility of this treatment following a washout period of 15 days. Both glands demonstrated desensitization of the isoproterenol-stimulated activity, and in the parotid gland treatment with DMI decreased the post-receptor signal transduction as well. The washout period of 15 days completely reversed the desensitization in the parotid gland and partially reversed the effects in the submandibular gland. These data suggest that desipramine-induced attenuation of β-adrenergic signal transduction is not limited to the brain and that these direct effects on salivary glands may explain the salivary dysfunction observed after administration of desipramine.

Relationship between cAMP production and protein secretion in rat submandibular acini: Evidence for loss of reserve capacity with age

We assessed isoproterenol and forskolin-stimulated cAMP production and protein secretion in submandibular salivary glands from young (4-7 mo) and old (25 mo) male F-344 rats. In senescent acini, the maximum amount of cAMP produced was three-fold less and the protein secreted was one-third less in response to the highest dose of isoproterenol (100 microM). Following isoproterenol stimulation, the young acini were more sensitive than the old for protein secretion than for cAMP production. Post-receptor stimulation (by forskolin) of cAMP production and protein secretion were less in the senescent rats. This report demonstrates the validity of an in vitro model for simultaneous assessment of biochemical and functional correlates. Furthermore, the observations suggest that both receptor and post-receptor stimulation of cAMP production and protein secretion diminish with age. Moreover, there is a reserve capacity for cAMP production with respect to protein secretion in the young that is diminished or even absent in the old rats.