Sex Differences in Renal Inflammation and Injury in High-Fat Diet-Fed Dahl Salt-Sensitive Rats

Roxanne Fernandes, Hannah Garver, Jack R. Harkema, James J. Galligan, Gregory D. Fink, Hui Xu (Hypertension, 2018)

High fat diets (HFD) are known to lead to obesity, hypertension, and renal dysfunction. A HFD can also lead to an increase in sympathetic outflow, which is the main cause of hypertension. Hypertension is chronically high blood pressure and is one of the major causes of renal disease. Although sex differences in obesity and cardiovascular disease have been researched, it is not known how a HFD causing hypertension and renal injury differ between males and females. The goal of this study is to see if there are differences in blood pressure, renal injury, and fat accumulation between sexes.

This study used HFD Dahl SS rats that exhibit signs of HFD-induced hypertension and Sprague Dawley rats that are also fed HFD but are not hypertensive. Beginning at three weeks old, the rats were either given a control diet that had normal salt levels or a HFD. The Dahl SS rats were given the HFD for either 10, 17, or 24 weeks, while the Sprague Dawley rats were placed on the HFD for 24 weeks.

After 24 weeks HFD males and females exhibited an increase in adipose tissue and body weight than the control rats. Body weights remained similar in all groups through week 8 of the diet, but then the control and the HFD males body weight increased significantly more than the females. Leptin, which is produced by adipose cells, was also shown to increase in males and females that were fed the HFD. Rats given the HFD also exhibited increased blood pressure. After 10 weeks of the diets, there was no significant difference between HFD and control rats. At 15 weeks the blood pressure of HFD rats became significantly higher than the controls rats. From 15 weeks to 24 weeks blood pressure in the HFD rats continued to significantly increase compared to the control rats. The heart rates between the HFD and control rats did not differ throughout the 24 week experiment. 

At 24 weeks, rats were given an injection of hexamethonium, which is a ganglionic blocker for sympathetic nerve activity. The hex injection decreased blood pressure more in the HFD males than the control males. The female rats exhibited a larger blood pressure decrease in the control rats than the HFD rats. When comparing males and females, the HFD males exhibited a larger blood pressure decrease compared to the HFD females. 

Researchers also observed renal histological changes. At 10 weeks, male rats had higher renal injury scores than females in both the HFD and control groups. At 17 weeks, male rats given the HDF exhibited significantly higher renal injury scores than the HFD females or the control groups. At 24 weeks, the HFD males exhibited an increased injury score compared to 17 weeks. This time period was also when blood pressure in HFD males and females was significantly increased.

Researchers then looked at the renal inflammatory responses in HFD rats. At 10 weeks, cortical and medullary macrophage infiltration was higher in males than females. At 17 weeks, macrophage levels increased in females of both diets, while males stayed similar to 10 week males. At 24 weeks, female rats exhibited a decrease in macrophages when compared to 17 week females, but still higher than 10 week females. While macrophage levels in males at 24 weeks was similar to the males at 10 and 17 weeks, levels were overall higher than female macrophage levels. The levels of cortical and medullary T-cells were then measured. At 10 weeks, all male and females had lower levels of T-cells. T-cells increased at 17 weeks through 24 weeks in males but not females, which exhibited similar T-cells levels to 17 weeks. Levels of inflammatory cytokines did not exhibit a significant change between different diets.

In conclusion, the HFD males and females both exhibited an increase in adipose tissue compared to the controls, but males generally have a higher body weight. Male and female rats given the HFD exhibited increase blood pressure, while heart rate shoed no significant difference between groups. The ganglionic blocker hexamethonium lead to a larger decrease in blood pressure in male rats compared to female rats with HFDs. Renal injury increased over the course of 24 weeks in males, while renal injury in females remained lower over the 24 week experiment. Renal T-cell levels increased over 24 in both HFD and control male rats, while females did not. 

One of the major limitations of this study that researchers discussed is how sex hormones effect hypertension and organ damage. They discuss that future studies could observe how sex hormones and receptors play a role in sex differences in hypertension. Another limitation discussed by researchers was how the HFD caused fat accumulation. Future studies could show that other high calorie diets can dramatically change renal inflammation or hypertension. I chose this paper because Dr. Fink recently spoke to the department and although his research is different than what we are doing in lab, it is also very closely related. Dr. Fink’s lab is mostly focusing on the sedentary side of our research and is now beginning to look towards how sex hormones are affecting hypertension.

-Paul M.

Microglia in the RVLM of SHR have reduced P2Y12R and CX3CR1 expression, shorter processes, and lower cell density

E. Myfanwy Cohen, Suja Mohammed, Mary Kavurma, Polina E. Nedoboy, Sian Cartland, Melissa M.J. Farnham, Paul M. Pilowsky. Autonomic Neuroscience: Basic and Clinical (2019).

Glial cells are cells of the central and peripheral nervous system that are not neurons. One type of glial cell, called microglia, act as a clean-up crew for the central nervous system. Microglia are macrophages that maintain the brain by looking for damage to the neurons or infectious chemicals. Since microglia look for damaged neurons, they also regulate inflammation in the brain. This study focuses on the microglia within a part of the brainstem called the rostral ventrolateral medulla (RVLM), which contributes to the control of blood pressure. The goal of the study was to determine if chronic high blood pressure is associated with a decrease in microglia function in the RVLM.

Fifteen week old spontaneously hypertensive rats (SHRs) and wild type rats were used in this experiment. Blood pressure was taken using a tail cuff to determine if the rats were hypertensive. The brainstems were removed and then placed in the freezer. The frozen brainstems were sectioned and the RVLM and the facial nucleus were punched out. Anti-Iba1 and donkey anti-rabbit AlexaFluor488 were used in the fluorescent tests. At the end of the experiment, rats were euthanized with sodium pentobarbitone.

A significant difference in systolic blood pressure was measured to demonstrate the two distinct groups. The SHRs exhibited a significant higher blood pressure (195 ±8 mmHg) than the wild type rats (144 ±8 mmHg). Two G-coupled protein receptors involved in the normal function of microglia, P2Y12R and CX3CR1, were measured and compared between the two groups of rats.  In the RVLM, P2Y12R expression was significantly lower in SHRs by about 37% when compared to the wild type rats. Expression of P2Y12R was also measured in the facial nucleus and exhibited no significant difference between SHRs and the wild type rats. Expression of CX3CR1 was then measured in the RVLM and the facial nucleus. In the RVLM, CX3CR1 expression was shown to be 30.9% lower in the SHRs when compared to the wild type. Expression of CX3CR1 in the facial nucleus was not significantly different between SHRs and the wild type.

The enzyme phenylethanolamine N-methyltransferase (PNMT) is found in the adrenal medulla and plays a role in converting noradrenaline to adrenaline. This allows PNMT to be a marker for adrenergic neurons, which are found in the RVLM but not the facial nucleus. The expression of PNMT was then compared between SHRs and wild type rats. The RVLM exhibited a significant amount of PNMT mRNA, while the facial nucleus showed a very small amount, which verifies the tissue punches have the correct sites.

Researches then observed the differences in microglia cell density between SHRs and wild types rats. The SHRs exhibited 22.9% lower cell density than the wild type. To further observe how active the microglia were, branch length, endpoints, and branch number were also examined. While the number of endpoints and branch numbers did not show a significant difference, branch length was significantly lower in the SHRs when compared to the wild type.

In conclusion, the G-coupled receptors CX3CR1 and P2Y12R play a major role in the normal function of microglia. Spontaneously hypertensive rats exhibit decreased expression of P2Y12R which may lead to the decreased microglia cell density in the RVLM. The researchers do state in the article that more research needs to be done to determine if the change in microglia are the cause or effect of over-activation of the RVLM and exactly how the microglia are being affected. I found this article interesting to our lab work because we constantly discuss the increased activation of RVLM but may not think about the exact repair mechanisms involved that may also not be working properly.

Differential activation of adrenal, renal, and lumbar sympathetic nerves following stimulation of the rostral ventrolateral medulla of the rat

Patrick J. Mueller, Nicholas A. Mischel, Tadeusz J. Scislo.

American Journal of Physiology – Regulatory, Integrative and Comparative Physiology (2011)

The sympathetic nerves are part of the autonomic nervous system, which controls unconscious actions, and are important in the response to changes in blood pressure.  The rostral ventrolateral medulla (RVLM) has been shown to direct sympathetic nerve activity (SNA) to cardiovascular targets and may have a role in the increased SNA of cardiovascular diseases. This study specifically looks at three areas of SNA: preganglionic adrenal SNA (ASNA), renal SNA (RSNA), and lumbar SNA (LSNA). The goal of this study is to determine how stimulating the RVLM effects the adrenal, renal, and lumbar sympathetic nerves individually.

This study used seventeen Sprague-Dawley rats and they were anesthetized with a mixture of alpha-chloralose and urethane. Microinjections into the RVLM were done by using a triple-barrel micropipette at a 90 degree angle. Dye was injected after the experiments were done to later identify the RVLM as the injection point. All rats were then killed using Fatal-Plus euthanasia solution.

Different concentrations of glutamate, which is the major excitatory neurotransmitter, were injected to observe responses in nerve activity, blood pressure, and heart rate. When the different concentrations were given in a fixed volume, the blood pressure was shown to be significantly increased and while there was a small increase in heart rate, it was not significant. The preganglionic ASNA was shown to significantly increase at each concentration of glutamate. The 10mM and the 100mM concentrations of glutamate did not produce a significant difference in RSNA, but they were significantly increased from the 1mM concentration change in nerve activity. LSNA was significantly increased after the 100mM glutamate injection when compared to the 1mM or 10 mM concentrations. The preganglionic ASNA was shown to exhibit significant increased activity as the concentrations were increased. Overall, the preganglionic ASNA activity exhibited the largest increase out of the three nerves.

The next test was to keep the concentration fixed but change the volume of glutamate. Volumes of 15, 30, 60, or 90 nl were injected into the RVLM and then changes in nerve activity, heart rate, and blood pressure were observed. While heart rate was shown to not significantly change, blood pressure significantly increased at a volume of 90 nl when compared to 15 nl. Nerve activity did increase, but not as large of an increase as when volume was fixed and concentration increased. When the volume was changed from 15 nl to 30 nl, the preganglionic ASNA was the only one to show a significant increase. At 60 nl the RSNA was significantly increased compared to the activity at 15 nl. Nerve activity at 90 nl of glutamate exhibited increases in both RSNA and preganglionic ASNA, but not LSNA. While there did seem to be a small increase in LSNA, it was not significant.

Another study compared nerve activity after given a dose of sodium nitroprusside (SNP) to the nerve activity after a bicuculline (Bic) injection. SNP lowers blood pressure to cause an increase in nerve activity, which attempts to counteract the dilating effects of SNP. Bic is a GABA receptor blocker which leads to a large increase in nerve activity.  The increase in nerve activity cause by Bic was about four times greater than the increase in nerve acitivty caused by SNP lowering blood pressure. While all nerves exhibited an increased response, preganglionic ASNA had the largest increase in response to SNP and Bic.

In conclusion, all three nerves exhibited increased sympathetic nerve activity when the RVLM was stimulated, though preganglionic ASNA increased more RSNA and LSNA. When the RVLM inhibitor is blocked, the SNA increase is much higher that the natural increase during low blood pressure. What I found most interesting about this study was the difference in activation between the Bic and SNP injections. I had not realized how much greater the response would be if all GABA receptors are blocked. This data helps to reinforce that even if blood pressure is very low and nerve activity is increasing to respond, there are still inhibitory factors in play.

-Paul M

GABAB receptor-mediated mechanisms in the RVLM studied by microinjections of two GABAB receptor antagonists

GIAN LUIGI AVANZINO, PIER0 RUGGERI, DONATELLA BLANCHI, CARLA E. COGO, ROSA ERMIRIO, AND LYNNE C. WEAVER
Istituto di Fisiologia Umana, Universith degli Studi, Viale Benedetto XV 3, I-l 6132 Genoa, Italy; and The John P. Robarts Research Institute, London, Ontario N6A 5K8, Canada
Journal of American Physiology Society (1994)

The current study is one of the first to investigate the role of GABA_B receptors in the RVLM. While it is older than others, the study is important to understand the history of the microinjection technique and the previous investigations of the GABA receptors in the RVLM. At the time of the study, the GABA_B antagonists (2-OH-s and CGP-35348) had just been created, allowing the study to research whether or not the GABA_B receptors were present in the rostral ventrolateral medulla (RVLM). Additionally, Avanzino et al. wanted to determine whether or not GABA_B receptors contribute to the regulation of the sympathetic nervous system output (which the researchers call the central cardiovascular regulation).

The animals were paralyzed and artificially respirated after the first round of microinjections to control for any respiratory changes during the measurements. Heart rate and blood pressure were measured during the microinjection experiments. The researchers assumed that if the GABA_B receptors were present in the RVLM, the antagonists would produce an increase in the blood pressure and heart rate. Two different microinjections followed: 1. Rats received bilateral microinjections of one of the GABA_B receptor antagonists or; 2. Rats received unilateral microinjections of the antagonist followed by either a GABA_A or GABA_B receptor agonists following the electrolytic lesion of the contralateral RVLM.

The first experiments produced increases in blood pressure and heart rate after the microinjections of either of the GABA_B receptor antagonists. These results held true after the rats were paralyzed, as well. The researchers concluded that the GABA_B receptors could be present in the RVLM and may contribute to the regulation of the central cardiovascular regulation. To further investigate this, the researchers injected the antagonists with either a GABA_B agonist or a GABA_A agonist. During this second experiment, the microinjections were completed unilaterally after the electrolytic lesion of the opposite RVLM. After the injections of 2-OH-s and CGP-35348, either badlofen (the GABA_B agonist) or muscimol (the GABA_A agonist) followed. Both of these agonists produced depressor responses when injected alone. However, the antagonists prevented the decrease in blood pressure only when injected with badlofen. The muscimol + antagonist injections were not significantly different to the muscimol alone. The same results were obtained in the paralyzed animals.

The results from the study suggested that GABA_B receptors are present in the RVLM in rats. These results are useful for future studies looking to further investigate the regulation of the RVLM in the central cardiovascular regulation. Additionally, the protocol of the experiment shows the significance of the microinjections. This experiment type is useful for studying the presence of receptors, and for investigating the function of brain regions, specifically the RVLM.

-LivInLaVida

Sympathoexcitation by hypothalamic paraventricular nucleus neurons projecting to the rostral ventrolateral medulla

By Satoshi Koba, Eri Hanai, Nao Kumada,  Naoya Kataoka, Kazuhiro Nakamura, and Tatsuo Watanabe 

Division of Integrative Physiology, Tottori University Faculty of Medicine, 86 Nishi-cho, Yonago, Tottori 683-8503, JapanDepartment of Integrative Physiology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan 
Journal of Physiology, 596.19 (2018) pp 4581–4595 

The rostral ventrolateral medulla (RVLM), a brain region involved in the baroreceptor reflex, is essential in the regulation of blood pressure through spinally projecting neurons that contribute to the sympathetic nervous output. The RVLM receives afferent input from the paraventricular nucleus (PVN), which is thought to regulate the outflow from the RVLM (PVN-RVLM neurons). However, the excitatory role of the PVN-RVLM neurons has never been researched prior to this study. Through the use of optogenetics, Koba et al. investigated the excitatory role of the PVN-RVLM neurons on the effects of renal sympathetic nerve activity (RSNA) and the coinciding changes in mean arterial pressure (MAP).

Three experiments were completed in order to answer the study’s question: 1. Photostimulation of PVN-RVLM axons on renal sympathetic nerve activity; 2. The effects of glutamate receptor blockade in the RVLM on the photostimulation of PVN and; 3. The effects of intermittent photostimulation of PVN-RVLM neuronal cell bodies on renal sympathetic nerve activity.  

The study used rats more than 7 weeks old to complete the experiments. To prepare the animals for the optogenetic studies, their PVN-RVLM neurons were first transfected with either a control virus vector (pAAV2-CMV-palGFP) or the channelrhodopsin-variant containing vector (pAAV2-CMV-ChIEF-tdTomato). The vectors were microinjected into either the PVN unilaterally or the RVLM bilaterally, depending on which experiment the animals were used for. Immunofluorescence staining determined which type of neuron was transfected after the experiment.

Experiment 1 animals received unilateral microinjections of the vectors unilaterally within the PVN. The experiment found that the active vector animals had significant increases in RSNA and MAP compared to baseline after photostimulation within the RVLM. Thus, the pre-synaptic PVN neurons expressed channelrhodopsin, which would activate the neurons at PNV-RVLM neurons, leading to excitation down to the renal sympathetic nerve. This did not happen in the control animals, signifying that they did not have channelrhodopsin expressed in the synapses of the PVN-RVLM animals. These neuron groups also expressed a large amount of VGLUT2 (vesicular glutamate transporter 2) in the PVN-derived axons, which lead the researchers to think that the glutamate transmission to the RVLM leads to excitation.

Experiment 2 investigated whether or not the PVN-RVLM neurons acted through glutamate to produce sympathoexcitation. Glutamate blockers AP5 and CNQX were unilaterally microinjected into the RVLM. After glutamate injection, the photostimulation did not produce the sympathoexcitation that was previously measured. Thus, the researchers concluded that glutamate was at least one of the neurotransmitters needed for sympathoexcitation with the PVN-RVLM neurons.

To further understand the how the PVN-RVLM neurons work, the cell bodies within the PVN received photostimulation, rather than the axons (Exp. 1). The cell bodies received 1-minute intervals of photostimulation at 10, 20, and 40 Hz, revealing a “synchronous” activation at the level of the renal sympathetic nerve. The level of activation correlated to the level of Hz used to stimulate the nerve.

The researchers concluded that the glutaminergic PVN-RVLM neurons help to drive sympathoexcitation. These neurons act on C1 neurons within the RVLM, which then project down the spinal cord to modulate blood pressure in the rats. While this study was interesting, it stuggled with transfecting all of the animals. Therefore,  it is difficult to say that all possible PVN-RVLM neurons were correctly transfected, which may produce inaccurate results otherwise. More research should be done of the efficacy of the optogenetic methods used within this paper. Furthermore, the role of non-C1 neurons, which also receive PVN input, may have been activated as well. Their activation may play a role in sympathoexcitation, and more research should be done. It would also be interesting to see the effects in rats younger than 7 weeks old. Additionally, female rats should be investigated, due to effects that estrogen may have on baroreceptor reflex. 

-LivInLaVida