Construction of time-response curve for neuronal and vascular endothelial dysfunction in ovariectomized rats.

BACKGROUND: Studies have shown that there is a critical time period to start hormone therapy after the loss of ovarian function during menopause. The length of estrogen deprivation may evolve different pathophysiological manifestations.
OBJECTIVE: The aim of the present study was to investigate behavioral, biochemical, and molecular alterations at different time points after surgical menopause with an aim and identify various pathophysiological targets to exploit "window of opportunity" and to design newer therapeutic modalities for menopause-associated neurobehavioral and vascular deficits.
MATERIALS AND METHODS: Bilateral ovariectomy was performed to induce surgical menopause and estrogen deficiency state. Menopause-associated neuronal and vascular dysfunctions were noted after 1, 2, and 3 months of the study.
RESULTS: Neuronal and vascular endothelial dysfunction post ovariectomy revealed that behavioral, biochemical, molecular, and vascular endothelial dysfunction appeared after 1 month of ovariectomy except hyperglycemia, which occurs after 3 months.
CONCLUSIONS: Time-response studies measuring behavioral, biochemical, and molecular markers at various time points after ovariectomy reveal that there is a fast onset of neuronal and vascular complications, but the duration of insulin resistance is a relatively late phenomenon.

Introduction

Ovariectomy in rodents is a frequently used animal model to study menopause-induced diverse pathologies that result from the precipitous fall in circulating estradiol levels. Although a significant amount of research data has been generated from this model, literature is replete with reports that the timing after estrogen decline, either surgical or natural, is an important determinant of onset of postmenopausal neurological disorders. Abrupt oscillations in gonadal hormones such as estradiol, follicle-stimulating hormone (FSH), and luteinizing hormone lead to variable periods of vulnerability to brain disorders.[1] The endocrine milieu strongly influences the sensitivity to serotonergic and noradrenergic systems. Hormonal oscillations due to removal of ovaries as well as by exogenous estradiol, have been demonstrated to modulate serotonin transporters as well as sensitivity to 5-HT1A receptors.[2]

Estrogen deficiency-induced inflammatory response plays an important role in painful pathological conditions as it is reported that proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, and IL-16) either directly stimulate nociceptors or modulate the release of bradykinin and substance P. Second, brain-derived neurotrophic factor (BDNF) acts as a neuromodulator in nociceptive signaling. Thus, increased pain sensitivity after menopause may be due to a fall of BDNF levels in the nociceptive sensory pathway, as it is reported that estrogen regulates growth factors such as BDNF mRNA levels and nerve growth factors in the central systems associated with nociception.[34]

Estrogen deficiency increases the risk of cognitive deficits and extensive literature reports suggest that estrogen replacement therapies can be protective against cognitive deficits. Estrogen can encourage nerve growth processes and modulate synaptic plasticity.[5] Forebrain cholinergic neurons express estrogen receptors (ERs), and estrogen supplementation enhances cholinergic function after ovariectomy.[6]

Along with neurological disorders, estrogen deficiency also leads to vascular endothelial dysfunction and metabolic disturbances such as increased body weight, dyslipidemia, hyperglycemia, insulin resistance, proinflammatory states, and increased risk of heart diseases and diabetes mellitus.[78]

All the above findings show that the absence of ovarian hormones has a pivotal role in neurological, metabolic, and vascular disorders; however, literature reports on the duration of hormone deprivation and the onset of these deficits were very contradictory. Thus, the main objective of the present study was to determine the time course of behavioral (memory, depression, pain), biochemical, molecular, and vascular endothelial changes in ovariectomized (OVX) rats.

Materials and Methods

Young female (200–250 g), 3–6 months old Sprague Dawley rats, were obtained from Panjab University central animal house and maintained on natural light and dark cycle and had free access to food and water. All the animal experiments were performed according to policies of the institutional animal ethics committee (approval no. IAEC/282; dated: 30-08-2012).

Experimental design

To evaluate behavioral, biochemical, molecular, and endothelial alterations at different time points after surgical menopause, SD rats were divided randomly into five groups (n = 6–8): control group, sham-operated, 1-month, 2-month, and 3-month OVX rats.

Bilateral ovariectomy in rodents is the most extensively used animal model of surgical menopause induced. To perform bilateral ovariectomy, we followed a double dorsolateral approach as used earlier by Park et al.[9] Basic physiological parameters such as body weight, food, and water intake were measured every week during the study protocol using metabolic cages. Menopause-associated neuronal dysfunction was noted after 1, 2, and 3 months to study time-dependent changes in three neurobehavioral paradigms, i.e., pain, depression, and memory. Memory functions (escape latency, pathlength, total time spent in target quadrant [TSTQ], frequency, and percentage of total path length traversed in target quadrant) were assessed using a computer tracking system (EthoVision software) via Morris water maze test.[10] Immobility time, i.e., measurement of depression, was assessed by the original method of Porsolt et al.,[11] using forced swim test. Mechanical hyperalgesia, tactile allodynia, and thermal hyperalgesia were assessed using Randall–Selitto analgesiometer,[12] von Frey Hair Test,[13] and tail immersion test[14] correspondingly.

After that, animals were sacrificed, their uterine horns were isolated and weighed. Serum estradiol levels were monitored quantitatively according to the manufacturer's (DRG® International, USA) instruction using a commercial ELISA kit. Vascular functions were assessed in isolated rat thoracic aorta. In this, endothelium-dependent ACh-induced relaxation and endothelium-independent sodium nitroprusside (SNP)-induced relaxation were measured.[15] The integrity of vascular endothelial lining was assessed by an electron microscopic study.[16]

Plasma glucose levels and lipid profile were estimated by commercially available spectrophotometric kits (Erba glucose kit, India). Plasma insulin levels were assessed using ELISA Kit by DRG International, Inc., USA. The malondialdehyde content,[17] reduced glutathione (GSH),[18] superoxide dismutase (SOD) activity,[19] nitrite levels,[20] and acetylcholinesterase levels were estimated in whole brain.[21] The brain and serum TNF-α and IL-1β were quantified by R&D Systems ELISA kits [Figure 1].

Figure 1

Experimental protocol

Statistical analysis

Statistical analysis was performed by means of GraphPad Prism 376R statistical software Sandiego, CA, USA. Data were expressed as mean ± standard error of mean and P < 0.05, 0.01, and 0.001 were considered significant for group difference. Data for escape latency, pathlength, and ACh-induced endothelium-dependent relaxation were statistically analyzed using two-way ANOVA. However, the remaining parameters were evaluated by one-way ANOVA followed by Tukey multiple range test.

Results

Time-course of changes of basic physiological parameters lipid and glycemic profile after 1, 2, and 3 months of ovariectomy

All OVX rats exhibited significant (P < 0.05) reduction in circulating estradiol levels and uterine weight after 1 month (7.7 ± 2.1 and 105.5 ± 7.1), 2 months (6.4 ± 1.2 and 93.2 ± 11.5), and 3 months (6.2 ± 1.4 and 84.6 ± 9.2) of ovariectomy as compared to sham group rats (56.9 ± 1.5 and 452.5 ± 16.3), thereby confirming the menopausal state. % age change in body weight was significantly increased and after 1, 2, and 3 months (20.6%, 22.9%, and 23.5%) of ovariectomy when compared to the sham group (9.4%). However, no significant amend in water and food ingestion was noted in OVX versus sham group rats. Blood cholesterol levels and triglyceride levels were significantly increased, while HDL levels were significantly decreased after 1 month (112.7 ± 6.4, 44.1 ± 3.8, and 36.1 ± 1.6), 2 months (137.9 ± 2.8, 61.6 ± 3.2, and 30.2 ± 3.2), and 3 months (192.2 ± 2.7, 69.7 ± 2.4, and 26.5 ± 3.2) of ovariectomy as compared to sham group rats (82.3 ± 3.8, 34.1 ± 4.6, and 46.3 ± 1.2), respectively. The blood glucose levels (1.2, 1.4, and 2 fold), insulin levels (1.2, 1.3, and 1.4 fold), and hemoglobin A1c levels (1.2, 1.3, and 1.4 fold) also increased time dependently after 1, 2, and 3 months of ovariectomy as compared to sham group rats, but significant increase in blood glucose levels was observed only after 3 months of ovariectomy [Figure 2].

Figure 2

Effect of ovariectomy on plasma glucose (a), glycosylated hemoglobin A1c (HbA1c, %) (b), and plasma insulin levels (c) after 1, 2, and 3 months of ovariectomy. Data are expressed as mean ± standard error of mean S.E.M. (*) P < 0.05 versus sham; OVX = Ovariectomy

Neurobehavioral parameters

Effect of ovariectomy on memory in Morris water maze after 1, 2, and 3 months of ovariectomy

A significant (P < 0.05) increase in escape latency and pathlength was noted after 1, 2, and 3 months of ovariectomy when compared to sham group on 5th day trials [Figure 2]. The TSTQ was significantly lowered in OVX after 1, 2, and 3 months of ovariectomy (1.5, 1.6, and 2 fold) with respect to sham. Similarly, in the probe trial, the frequency of appearance in the target quadrant was significantly decreased after 1, 2, and 3 months of ovariectomy (11.9 ± 0.4; 9.3 ± 0.7; 8.4 ± 0.6) versus sham group (16.3 ± 0.5) [Figure 3]. Significant decrease in percentage of total pathlength traversed in the target quadrant was observed after 1, 2, and 3 months of ovariectomy as compared to sham group rats [Figure 3].

Figure 3

Time-dependent changes on escape latency (a) and pathlength (b) time spent in the target quadrant (c) and frequency of appearance in the target quadrant (d) and percentage of total pathlength traversed in target quadrant (e) using Morris water maze after 1, 2, and 3 months of ovariectomy. Values are expressed as mean ± standard error of mean. (*) P < 0.05 versus sham

Time course of changes on immobility time after 1, 2, and 3 months of ovariectomy using forced swim test

The mean immobility period was significantly increased after 1 month of ovariectomy (90.6 ± 12.1) as compared to sham group rats (60.1 ± 4.9) which marginally decreased after 2 and 3 months (86.2 ± 17.2 and 83.9 ± 2.6.), but the decrease was not statistically significant [Figure 4].

Figure 4

Time-dependent changes on immobility time using forced swim test after 1, 2, and 3 months of ovariectomy. Values are expressed as mean ± standard error of mean. (*) P < 0.05 versus sham

Time course of changes on pain parameters after 1, 2, and 3 months of ovariectomy

In Randal–Selitto test, OVX rats showed (36.3, 33.7, and 34.7%) a decrease in paw withdrawal threshold after 1, 2, and 3 months of ovariectomy as compared to sham group rats [Figure 5]. Increase in pain sensitivity in von Frey Hair Test after 2 and 3 months of ovariectomy was 1.39 and 1.44 fold as compared to the sham group; however, no significant results were observed after 1 month of ovariectomy. Tail flick latency was significantly decreased after 1, 2, and 3 months of ovariectomy (P < 0.05) [Figure 5]. However, no difference was observed on mechanical hyperalgesia, mechanical allodynia, and thermal hyperalgesia in sham and control group rats.

Figure 5

Effect of ovariectomy on mechanical hyperalgesia (a), mechanical allodynia (b), and thermal hyperalgesia (c) after 1, 2, and 3 months of ovariectomy. Values are expressed as mean ± standard error of mean. (*) P < 0.05 versus sham

Biochemical and molecular estimations

Significant increase in thiobarbituric acid and reactive substances levels was observed (P < 0.05) in the brain of OVX rats after 1, 2, and 3 months of ovariectomy [Figure 6]. Along with this, SOD and reduced GSH levels were significantly reduced in OVX after 1, 2, and 3 months of ovariectomy (P < 0.05) [Figure 6]. Brain acetylcholinesterase activity and nitrite levels were significantly increased time dependently after 1, 2, and 3 months of ovariectomy [Figure 7]. Inflammatory markers such as TNF-α (1.5 times) and IL-1β (1.5, 1.8, and 1.9 times) were increased after 1, 2, and 3 months of ovariectomy, respectively [Figure 7].

Figure 6

Effect of ovariectomy on brain TBARS levels (a), superoxide dismutase levels (b), glutathione levels (c), nitric oxide levels (d) and acetylcholinesterase levels (e) after 1, ,2 and 3 months of ovariectomy. Values are expressed as mean ± standard error of mean. (*) P < 0.05 versus sham

Figure 7

Effect of ovariectomy on brain tumor necrosis factor alpha (a) and interleukin-1β levels (b) after 1, 2, and 3 months of ovariectomy. Values are expressed as mean ± standard error of mean. (*) P < 0.05 versus sham

Effect of ovariectomy on vascular parameters after 1, 2, and 3 months of ovariectomy

ACh- induced relaxation was significantly attenuated in OVX rats after 1, 2, and 3 months of ovariectomy [Figure 8]. However, no difference was observed in SNP-induced relaxation [Figure 8]. Further, oxidative stress marker (TBARS) levels and inflammatory marker (TNF-α and IL-1β) levels were significantly increased in serum (P < 0.05) after 1, 2, and 3 months of ovariectomy as compared to sham group rats time dependently [Figure 9].

Figure 8

Effect of ovariectomy on acetylcholine-induced endothelium-dependent relaxation (a) and sodium nitroprusside-induced endothelium-independent relaxation on isolated aortic ring preparation (b) after 1, 2, and 3 months. Responses are expressed as percentage of maximum contraction induced by phenylephrine (3 × 10−6 M). Data are expressed as mean ± standard error of mean. (*) P < 0.05 versus sham

Figure 9

Time course of changes on serum TBARS levels (a), tumor necrosis factor-α (b), and interleukin-1β (c) levels after 1, 2, and 3 months of ovariectomy. Data are expressed as mean ± standard error of mean. (*) P < 0.05 versus sham

Discussion

Effect of ovariectomy on behavioral (depression, pain, and memory), biochemical, and molecular parameters after 1, 2, and 3 months

Various studies have shown that menopause leads to an increased incidence of depression. Although the mechanisms via which ovarian hormone alterations lead to the development of depression are not clear, estrogen withdrawal theory suggests that reduction in gonadal hormone during the transition of menopause exacerbates depressive mood.[22] Accumulating evidence showed that ERs are situated in mood and behavior brain areas and regulate serotonergic and noradrenergic neurotransmission. Estrogen insufficiency selectively reduces serotonin receptor expression in the hypothalamus and the preoptic area of OVX rats which can be reversed by treatment with estrogen.[22] Accordingly, in our study significant reduction in serum estradiol levels and increased immobility time after 1, 2 and 3 months of ovariectomy. Estrada-Camarena et al.,[23] was reported that female Wistar rats showed immobility after 1 week of ovariectomy but not at 3 and 12 weeks. Li et al.[24] reported that depression occurs in female SD rats after the 5th week of ovariectomy. In agreement with this, we also observed a significant increase in depression after 1 month of ovariectomy, which was consequently decreased but not at a significant level after 2 and 3 months. A possible explanation behind this may be an increase in FSH levels during the menopause transition. Studies have reported that an increase in FSH is positively correlated with depression scores in perimenopausal women. A couple of studies on rats also reported that FSH levels in rats start increasing after 2 days of ovariectomy and it persists for at least 1 month after ovariectomy. Carretti et al.[25] reported that levels of gonadotrophin hormone impact serotonergic function, i.e., FSH levels are inversely related to serotonin release. Our results are in concurrence with studies reporting that in early menopause, depressive symptoms are more recurrent than late menopause. Feld et al.[26] reported that mood changes are observed transitionally in menopause syndrome and not always persistent during menopause. Moreover, an adaptive process may set, after a long-time absence of ovarian fluctuations, which leads to normal behavioral effects.

Sex hormones play a key role in pain perception. Various studies reported that estrogen plays a key role in pain modulation. Allen and McCarson[3] reported that estrogen deficiency reduces the release of growth factors playing a neuromodulatory role in nociceptive signaling. Further, estrogen deficiency also increases the release of inflammatory cytokines, which excite nociceptors either directly or stimulate the release of bradykinin and substance P. A study by Li et al.[24] reported that thermal hyperalgesia starts at the 2nd week of ovariectomy and persists for 7 weeks. However, mechanical allodynia occurs after 5 weeks of ovariectomy, which indicates that estrogen deprivation after menopause effects differentially thermal and mechanical nociception. We also observed in our study the induction of nociceptive hypersensitivity characterized by mechanical allodynia, hyperalgesia, and thermal hyperalgesia in the tail after 1, 2, and 3 months of bilateral ovariectomy. The onset of pain hypersensitivity occurred at 1 month after ovariectomy and it persisted till 3 months. These behavioral pain parameters were further correlated with increased TNF-α and IL-1β levels and oxidative stress markers in the brains of OVX rats. These findings indicate that even short-term estrogen depletion results in inflammation and oxidative surge resulting in postmenopausal hyperalgesia.

Preclinical and clinical evidences showed that loss of ovarian hormone resulting from surgical menopause causes impairment of learning and memory which can be reversed after treatment with estrogen replacement therapies. The protective effect of estrogen in memory is attributed to its potent cholinomimetic,[6] antioxidant, anti-inflammatory, antiapoptotic, and neurotrophic activity. Morris water maze is the most common method used for assessment of learning and memory.[10] In our study, the latency and distance to find hidden platforms were significantly increased, while TSTQ was markedly reduced after 1, 2, and 3 months of ovariectomy in SD rats. Previous studies have reported that changes in estrogen levels markedly affect cholinergic neurons.[6] The rise of inflammatory cytokines and oxidative stress also plays an important role in cognitive deficits. We also observed in our study a marginally time-dependent progressive increase in oxidative stress, inflammatory, and acetylcholinesterase levels which were correlated with cognitive dysfunction at various time intervals.

Effect of ovariectomy on body weight, glycemic profile, and lipid profile after 1, 2, and 3 months

Studies reported that the incidence of metabolic diseases such as obesity and diabetes mellitus is increased after menopause. Alterations in lipid metabolism and body fat distribution alterations are the major considerable factors which further increased insulin resistance syndrome prevalence after menopause.[27] In concordance with this, we also observed a significant increase in body weight and hyperlipidemia after 1, 2, and 3 months of ovariectomy. Glycemic profile was also impaired time dependently after 1, 2, and 3 months, but results were significant only after 3 months of ovariectomy. These results are in agreement with the previous report by MacDonald et al.[28] that body mass increases in a very short duration (1-week post ovariectomy), but glucose intolerance in these animals occurs only after 10 weeks. Further, several studies reported that in the OVX rodents, long-term ovariectomy decreases insulin sensitivity. Kim et al.[29] compared the long-term (10 weeks) and short-term (2 weeks) plasma glucose levels and reported that it did not change after 2 weeks. They suggested that the increase of insulin resistance after 10 weeks of ovariectomy occurred is not due to direct estrogen deficiency but is due to ovariectomy associated with obesity and estrogen deficiency both. Estrogenic effect to regulate pancreatic β-cell function has been well established.[78] It may be possible that after prolonged estrogen deficiency, pancreatic β-cells fail to compensate and result in impaired glycemic profile.

Effect of ovariectomy on vascular endothelial dysfunction after 1, 2, and 3 months

Estrogen is a key atheroprotective molecule with noticeable effects on the vasculature. Estrogen shows a beneficial effect on the vasculature either by directly acting on the vessels or indirectly by the modulation of factors (enhance the synthesis of nitric oxide [NO], modulate vascular tone, regulate expression of endothelial NO synthase [eNOS], and endothelin gene). Estrogen also seems to play the main role in vascular remodeling, inhibiting the innermost layer proliferation after injury and increasing contractile protein expression in the myocardium. Loss of estrogen after ovariectomy impairs vasorelaxation in response to ACh. This impairment in ACh-induced relaxation may be due to a decrease in eNOS expression along with NO bioavailability which can be restored by estradiol treatment. In agreement with this, we have also observed a significant decrease in ACh-induced relaxation after 1, 2, and 3 months of ovariectomy. Along with this, studies have reported that there is a sturdy correlation between the risk of endothelium dysfunction and inflammatory markers. Inflammation results in increased production of oxidative stress, which finally causes endothelial dysfunction.[30] In our study, we observed a significant rise in inflammatory markers (serum TNF-α and IL-1β) and serum TBARS levels after 1, 2, and 3 months of ovariectomy in our study.

Conclusions

Time-response studies measuring behavioral, biochemical, and molecular markers at various time points after ovariectomy reveal that there is a fast onset of neuronal and vascular complications, but the duration of insulin resistance is a relatively late phenomenon.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.