Estradiol and Tamoxifen Reverse Ovariectomy-Induced Physical Inactivity in Mice

Gorzek JF, Hendrickson KC, Forstner JP, Rixen JL, Moran AL, Lowe DA.

Medicine and Science in Sports and Exercise 2007

                Physical activity is crucial to maintaining a positive health and it has been shown that there are many positive adaptations in multiple different systems that can come from regular physical activity. These systems can include the cardiovascular system, immune system, musculature system and probably the nervous system as well. Many studies have shown that female rats consistently run more than their male counterparts. Furthermore, it has been shown that this difference in running is most likely due to the presence of female hormones produced in the ovaries. Something interesting to me is why are the females running more than the males. If it is the ovarian hormones, what are they doing to cause the female to run more? Is it affecting their motivation to run or their physical ability to run? And if it is the physical ability to run, is it in the nervous system of in the skeletal and musculature system? The purpose of this study was to assess the effect that ovariectomy and then estrogen replacement or tamoxifen replacement had on voluntary physical activity as well as body mass.

                There were two studies conducted in this paper. In the first study mice were assigned to either sham group, ovariectomy with estrogen group, or ovariectomy with placebo group. At week four (14 weeks old) surgeries were performed which were either bilateral ovariectomy or sham surgery. After the surgery and then a week recovery period mice were given access to a wheel to run on. Mice ran for 4 weeks before either estradiol or placebo replacement took place. Subcutaneous pellets of estradiol or placebo were placed under the skin of the mice and then the mice were allowed to run for another 4 weeks at which point they were sacrificed.

                In the second study, all mice were ovariectomized and then placed in one of three groups: estradiol replacement, tamoxifen replacement or placebo replacement. In this study surgeries were performed at the same time as the pellet placement and then the mice were given 3 days to recover and then put in their cages with wheels. At this point mice were allowed to run for 6 weeks and then sacrificed.

                All mice were kept on 12:12 light dark cycle. Surgeries were performed under anesthesia. Wheel running was assessed by mean weekly measures of distance.

                As expected, in the first study, the group of ovariectomy mice ran significantly less than the control sham group. The ovariectomy with estradiol group ran more similar to the placebo group until the replacement period at which point they increased their running significantly and ran more alike to the control group.  In the second study the data shows that all three groups increased their running as they acclimated to the wheel in the pre-surgery time frame. Once surgeries were performed the running of the placebo group declined significantly from before the surgery. The groups that were immediately treated with tamoxifen or estradiol maintained their running behavior more steadily, although there was some decline over time. The treatment groups with tamoxifen and estradiol ran significantly more than the placebo group, but the two treatment groups were not significant from one another.
                This study indicates that the decrease seen in physical activity in females after loss of ovarian hormones is indeed due to the loss of those ovarian hormones. Due to the use of tamoxifen in this study, it was also indicated that this hormone dependent change in running behavior is mediated by the estrogen receptor. The study also suggests that do to their measurements in body weight, hind limb weight, and heart muscle weight, it was more likely that the estrogenic compounds caused the mice to have more motivation to run rather than causing changes in their musculoskeletal system that allowed them to run more. Still to be uncovered is whether or not estrogenic compounds could cause differences In the brain that that provide the nutrition and blood for more exercise to occur – aka RVLM.

Ben R

Sympathoinhibition and it reversal by naloxone during hemorrhage

By: Hasser and Schadt (Am. J. Physiol. 262 (Regulatory Integrative Comp. Physiol. 31):R444-R451,1992)

When a hemorrhage occurs, there is a decrease in sympathetic nerve activity (SNA), plasma norepinephrine, and vascular resistance. In addition to these as a result, hypotension is also seen. This study focuses on naloxone (an opioid receptor antagonist) and how it reverses these events. They hypothesized that an increase in SNA was specific to naloxone and not to the pressor response. They also hypothesized that hemorrhagic hypotension is associated with an inhibition of RSNA (renal), and this inhibition is maintained in the absence of treatment. Lastly, they hypothesized that reinfusion of hemorrhaged blood will reverse sympathoinhibition, regardless of naloxone.

Animals were conscious during experiment. Catheters and electrodes were inserted to record blood pressure and SNA. Blood was drawn until BP fell under 40mmhg, and then 5ml were drawn thereafter. Naloxone was then injected while constant recordings were being done.

Following hemorrhage, BP and RSNA significantly decreased. When naloxone was injected, BP, RSNA, and HR were significantly increased, compared to the injection of saline. Also, when blood was reinfused, BP returned to normal but the SNA did not recover to normal.

We see that when BP decreases, we would expect SNA to spike to compensate for the decrease. Instead we saw that SNA actually went down after blood loss and BP drop. When naloxone was given, SNA went back up, followed by and increase in BP. This occurred even though blood volume was still low. This shows that BP decreases as a result of a decrease in SNA. When blood was reinfused, along with naloxone, there was still sympathoinhibition.

This study was vital in showing that even though blood loss occurs, the SNA levels did not increase, which is what we see when BP goes down. Hemorrhage follows a different mechanism and I am guessing the SNA goes down to prevent the heart from being over worked. We see how SNA has more of a control on BP, then BP has on SNA

-Tsetse Fly

Is the RVLM a key site for sex-related differences in blood pressure regulation? Focus on “Sex differences in angiotensin signaling in bulbospinal neurons in the rat rostral ventrolateral medulla,” by Wang et al.

By Roger A. L. Dampney 

Bosch Institute and School of Medical Sciences (Physiology), University of Sydney, Sydney, Australia American Journal of Physiology: Regulatory, Integrative, and Comparative Physiology, 2008

The rostral ventrolateral medulla (RVLM) is a brain region that is central to the maintenance and regulation of blood pressure. It drives the blood pressure changes through the use of the sympathetic nervous system. As the nerve outflow increases, so does the response in blood pressure. According to this paper, one of the most important influences on blood pressure is the action of angiotensin II (ANG II) on the RVLM. The neurons of the RVLM contain angiotensin receptors that have been shown to drive increases in blood pressure by activating NADPH oxidase to create reactive oxygen species (ROS). Thus, previous research suggests that ANG-II can lead to the development of hypertension by overproducing ROS in the RVLM neurons. To further support these claims, it would be beneficial if they author noted the effects of ANG-II receptor antagonists. If the antagonists reduced the SNA outflow and responding blood pressure, the importance of ANG-II in the alteration in blood pressure would be highlighted much more. Nevertheless, it does appear that the hormone contributes to the control of blood pressure in the RVLM.

Some strong evidence exists to support the importance of ANG II in the sex-differences of blood pressure. The researchers state that understanding the ANG II effects in males versus females would reveal one of the causes of sex differences in human hypertension. The paper notes that males respond to ANG II infusions with greater increases in blood pressure from sympathetic nerve outflow than females. ANG type 1 receptors (AT1) are expressed at a higher quantity compared to males in the RVLM. The NADPH subunit p47 is lower in females, however. Nevertheless, ROS production, which was used to measure neuron activation levels in dissociated neurons after ANG II application, was similar in both sexes. The author suggests that the higher amounts of AT1 but not p47 counterbalanced each other’s effects in females. This counterbalance would then produce similar responses in males and females. However, the use of a receptor antagonist here would provide further support for these claims. If the blocked receptor prevents increases in blood pressure, it would be harder to argue that other inputs may be contributing to the attenuated response seen in females. Circulating female hormones may be somewhat responsible for the response that is seen with ANG-II. They may be acting on another component of the ROS pathway, such as the p47 subunit expression, to alter the response seen in females. Therefore, the antagonism of the AT1 would be an avenue worth investigating. Additionally, studies should investigate the impact of the sex hormones has on the expression of the ANG-II component's genes. If estrogen impacts their expression, it could explain why the AT1 and p47 levels differed between the sexes. Nonetheless, there are measurable sex-differences within the ANG-II pathway that can be accounted for in the RVLM.

Additionally, the researchers found that, when acted upon by ANG-II, the L-type Ca2+ currents in female neurons were higher than males. These results were supported by an additional study that showed the amount and the sensitivity of the Ca2+ channels differed between the two sexes. Due to the similar ROS production in males and females, it is suggested that the activation of the Ca2+ channels is independent of the previously mentioned ANG-II/ROS pathway. This hypothesis is supported by the results of another study. When the ANG-II/ROs pathway was blocked and the L-type Ca2+ current was activated with ANG-II, females had a greater blood pressure response compared to the males--thus the ANG-II/ROS pathway was not necessary to activate the neurons located there. 

These results should raise some additional questions. Females typically have attenuated blood pressure responses when compared to males. Therefore, why would the female RVLM neurons have increased activation levels compared to males? Estrogen is finally introduced into the ANG-II pathway and its effects are measured. 17-estradiol produced a decrease in the L-type Ca2+ currents in the RVLM, suggesting that this is one of the mechanisms that lead to lower blood pressure changes in female rats. Thus, the paper suggests that the change in blood pressure would depend on the ratio of ANG-II and estrogen that is in circulation and acting on the RVLM. Additional studies should be done to consider the in vivo levels of both compounds in order to determine if this is indeed the case. 

The research that was reviewed above suggests that there are sex-related differences in the ANG-II pathway expressed within the RVLM. While more research should be done to further support the results presented here, ANG-II and its effects on the RVLM seem to play an important role in regulating blood pressure and possibly on the development of hypertension. 

-LivIn la Vida

Angiotensin-Converting Enzyme 2 in the Rostral Ventrolateral Medulla Regulates Cholinergic Signaling and Cardiovascular and Sympathetic Responses in Hypertensive Rats

Yu Deng, Xing Tan, Miao-Ling Li, Wei-Zhong Wang, Yang-Kai Wang. Neurosci Bull. 2018

The rostral ventrolateral medulla (RVLM) is an area in the brainstem that regulates sympathetic nerve activity associated with cardiovascular mechanisms.  Angiotensin is a hormone that causes vasoconstriction which leads to high blood pressure. This study focuses on the angiotensin-converting enzyme 2 (ACE2) that converts angiotensin into a much less reactive form of angiotensin. Therefore, ACE2 can contribute to the lowering of blood pressure and help therapeutically with hypertensive patients. This study examines how hypertension is affected by high levels of ACE2 in the RVLM.

Higher concentrations of ACh were observed in the RVLM of spontaneously hypertensive rats (SHRs) when compared to wild type rats and VAChT, a vesicular ACh transporter, levels were also shown to be increased in SHRs compared to wild type. Atropine was then injected into the RVLM to show that ACh is acting as a major neurotransmitter. Before the atropine was injected, SHRs exhibited higher blood pressures and heart rates than the wild type rats. The atropine injection caused a significantly larger decrease in blood pressure and nerve activity in the SHRs. Both the wild type and SHRs exhibited a very similar decrease in heart rate.

To deliver the human ACE2 into mice models, ACE2 lentiviruses were created. A lentivirus is a type of retrovirus which inserts a piece of DNA into the host’s genome. A control lentivirus was also created that has all the same sequences, but left out the one coding for the human ACE2. The lentiviruses were then injected directly into the RVLM. The results of the lentivirus injection showed that the expression of ACE2 was significantly higher in the Lenti-ACE2 SHRs compared to the Lenti-control SHRs four weeks after the injection. The Lenti-ACE2 SHRs exhibited decreased blood pressure and heart rate three weeks after the injection. Urine samples were taken to test the levels of norepinephrine (NE), which showed that the SHRs given Lenti-ACE2 exhibited a 72% decrease in NE compared to the SNRs given Lenti-control.

Concentrations of ACh in the extracellular fluid of the RVLM were then tested using microdialysis. Four weeks after the injection, SNRs given Lenti-ACE2 had decreased concentrations of ACh and VAChT, the vesicular ACh transporter. Atropine was also injected into the SHRs that received Lenti-control or Lenti-ACE2. Blood pressure and nerve activity was shown to have a smaller decrease in the Lenti-ACE2 rats, while heart rate remained very similar between both groups. 

In conclusion, increased levels of ACE2 in the RVLM were shown to decrease hypertensive effects in rats. High levels of ACh and the a ACh transporter were found in the SHRs, but when the ACE2 lentivirus was injected, ACh levels decreased along with blood pressure and nerve activity. Urine tests showed that NE also decreased in the rats given the ACE2 lentivirus, which shows there is less excitation releasing the neurotransmitter NE. The authors discussed how there has not been significant evidence that there is any change in the muscarinic receptors during hypertension and more research needs to be done.

Paul M

Estrous Correlated Modulations of Circadian and Ultradian Wheel-Running Activity Rhythms in LEW/Ztm Rats

Franziska Wollinik and Fred W. Turek

Physiology and Behavior – 1988

               

                Steroid hormones that are produced by females act to modulate the circadian rhythms of activity in female rats. This can occur on a daily basis. Furthermore, there are differences between males and females that are abolished when female are ovariectomized. This peaks interest in the question, what are the female hormones doing? The current study aimed to determine if the estrous cycle affected daily wheel-running activity in female rats.

                LEW/Ztm female rats were used in this study and were kept on a light dark cycle of 12:12. At 80 days of age all rats were placed in a cage with a wheel and at the beginning of the experiment half of the animals were blinded so they did not know whether it was dark or light. Estrous cycle was only monitored in three sighted and three blinded animals. For all animals the amount of wheel revolutions in a five minute period was measured and used to calculate the amount of activity. Out of the 20 animals used in the study there were a total of 17 animals that showed a consistent 4-5 day estrous cycle and it was these rats that were used in the analysis of the data.

                Rats showed increased levels of activity during the proestrus and estrus stages of the cycle and lower activities on the metestrus and diestrus stages. Measurements of daily activity and duration varied across the estrous cycle in the rats. This study showed that the biggest differences in the measurements of activity was consistently on the day of estrus. It was approximately two times higher than on the day of metestrus. The blinded rats also showed variations in activity based on the estrous cycle like the sighted animals. This study was important in showing that the voluntary wheel running activity of rats various with the estrous cycle as is supported by our data as well. In this study the day of estrus was shown to be the day of highest activity across both groups. This group suggests that it is estrogen and progesterone that modulate the rhythmic effects on activity seen in the estrous cycle. This current study was mostly focused on the effect that estrogen had on the circadian and ultradian rhythms. This is an interesting paper for sure because it does show that there are differences in the wheel running behavior based on the estrous cycle and it is suggested to be estrogen and progesterone. This can be the basis for further research in our lab, however, I feel there are definitely some methodology that needs to be reconsidered from this paper. Specifically, I am confused about how they determined the estrous cycle and feel that they were using some arbitrary parameters. Furthermore, this paper did not ovariectomize rats and replace with estrogen as the focus was the rhythms and not the wheel running, in this paper it seems the wheel running was just a way to measure rhythms.

Ben R

Daily voluntary exercise alters the cardiovascular response to hemorrhage in conscious male rats

By: Joslyn K. Ahlgren, Linda F. Hayward (Autonomic Neuroscience: Basic and Clinical 160 (2011) 42–52)

Blood loss is described by the response called hemorrhage (HEM). The first phase is called the "compensatory" phase, which consists of an increased heart rate (HR) and sympathetic activity. This is maintained until blood loss reaches 15-20% of total blood volume (TBV). Once blood volume drops past the critical point, the body goes into the "decompensatory" phase, where there is a decline in HR and AP. This will lead to increased levels of renin, vasopressin, and epinephrine. This leads to the "recovery" phase where sympathetic tone is restored. The longer the body is exposed to a hemorrhage, the harder the chance of recovery becomes.

Exercise has been shown to contribute to the cardiovascular system. Chronic exercise has been associated with enhanced health outcomes. It also affects areas of the brain that control AP and HR. These same regions of the brain have also been seen to contribute to the autonomic functions during HEM. The hypothesis was that exercise rats would display a change in autonomic responses to severe HEM. More specifically, he compensatory would be altered slightly, but the decompensatory phase would be significantly altered.

36 male rats were pair housed for 6 weeks in active (running wheel) or sedentary (no running) wheel conditions. The rats were at rest for 30-60 min where AP, MAP, and HR were recorded. Rats were then subjected to either a 30% TBV HEM over 15 min, sit quietly, or underwent baroreflex testing.

The results showed that following HEM, HR and MAP declined in both groups, but overall, SED animals had a greater drop in MAP and HR in response to HEM, than active animals. HR was also significantly lower in SEDs. Also, the HRV analysis revealed putative alterations in EX vs. SED .The results support previous reports of daily exercise modifying neuronal excitability.

When blood loss initially occurs, baroreflex adjustments occur to compensate for this loss. This study showed that during this phase, exercise significantly augmented the baroreflex increases in HR in response to the drop in AP. During the decompensatory phase, MAP was significantly higher at the end of HEM in exercise animals. At 60 min, post HEM, HR was significantly higher in EX. This shows that EX does indeed affect the recovery phase and can help increase survival rates. The results in the study also support that exercise training improves the cardiovascular activity during hypotensive periods.

This study relates to our lab by focusing on how exercise affects the body as well as the brains ability to recover from a hemorrhage. The SNS is vital to this process and contributes to the animals overall health. I would like to see how the RVLM specifically changes during times of blood loss. I would predict that it would fire at a greater rate once barorelex receptors sense blood volume decrease.


-Tsetse Fly

Activation of estrogen receptor B-dependent nitric oxide signalling mediates the hypotensive effects of estrogen in the rostral ventrolateral medulla of anesthetized rats

By C. Shih
Department of Pharmacy and Graduate Institution of Pharmaceutical Technology, Tajen University, Taiwan, the Republic of China
Journal of Biomedical Science, 2009

The purpose of this study was to better understand the actions of estrogen in the rostral ventrolateral medulla (RVLM). Specifically, the different estrogen receptors and their role in inducing its cardiovascular protective responses were investigated. Based on previous research, the study hypothesized that estrogen acts on the ERβ receptor to activate nitric oxide (NO) to inhibit sympathetic outflow from the RVLM.

So as to prevent an unexpected effect from circulating estrogen, the study used male rats. The animals received bilateral microinjections into the RVLM. They were not measuring the effects of estrogen on the activation of the RVLM, but rather how estrogen alone attenuates resting blood pressure. Therefore, the animals received microinjections of either: estradiol (E2β); a selective estrogen receptor alpha (ERα) agonist (PPT); an estrogen receptor beta (ERβ) agonist (DPN); a general ER antagonist (ICI 182780); an ERα antagonist (MPP); an ERβ antagonist (R,R-THC); a general NO synthase (NOS) inhibitor (SMT); an nNOS inhibitor (7-NI); an endothelial NOS (eNOS) inhibitor (L-NIO); or a transcription inhibitor (AMD). The transcription inhibitor was used to see if the activated ERs would mediate its actions through nontranscriptional methods, as studies have previously suggested.

The microinjections of E2β (0.5, 1, or 5 pmol) and not E2α produced a dose-dependent decrease in MAP and vasomotor outflow, with the higher doses producing a suppression that lasted longer (3-4 hours after the injection). E2α produced no changes from baseline measurements. When all three types of antagonists were given alone, no change was measured. However, when the ICI 182780 was given, the E2β-induced responses were reversed. Similarly, when the general ER antagonist was given before an E2β microinjection, the hypotension and decrease in vasomotor tone were prevented. Thus, the researchers suggested that E2β act on ERβ in the RVLM in order to produce its suppressive responses.

To further understand the selectivity of the estrogen in the RVLM, ERα and ERβ agonists were bilaterally injected. Only the ERβ agonist, DPN, produced the similar dose-related decreases in MAP and vasomotor output compared to the injections of E2β. ERα agonist DPN showed no change in the measurements. Again, these results suggested that estrogen acts on ERβ in the RVLM to produce its attenuation effects of the cardiovascular system.

The role of NO was further investigated. When the NOS inhibitors were coinjected bilaterally with E2β into the RVLM, there were no measured responses in MAP or vasomotor output. Interestingly, the general NOS inhibitor L-NAME prevented the E2β responses. Additionally, the iNOS inhibitor and not the nNOS or eNOS inhibitors prevented the responses as well. Therefore, the researchers suggested that the NO produced by iNOS is needed for E2β to produce its attenuation effects in the RVLM. The results are interesting because previous studies have shown that the NO produced by the eNOS is also important for the effects of E2β. However, these were studied within the PVN and suggests that the effects of estrogen in the brain are nucleus-specific.

Additionally, when coinjected with AMD and E2β, there was no change in the expected E2β depressive responses, suggesting that their hypothesis was correct: the effects of E2β on ERβ are mediated by nontranscriptional methods.

In summary, the study suggests that the E2β acts specifically in the ERβ in the RVLM to produce the inhibitory responses of the cardiovascular system. NO from the iNOS is suggested to contribute to the effects after the activation of the ERβ in the RVLM.

-LivInLaVida