Peripheral nociception associated with surgical incision elicits remote nonischemic cardioprotection via neurogenic activation of protein kinase c signaling

Jones, W. Keith, et al. "Peripheral nociception associated with surgical incision elicits remote nonischemic cardioprotection via neurogenic activation of protein kinase C signaling." Circulation 120.11 suppl 1 (2009): S1-S9.

Ischemia reperfusion injury associated with myocardial infarction is a major contributor to cardiovascular related death.  Unfortunately, the only treatment we have for cardiac ischemia, is reperfusion of oxygen to the ischemic tissue, which in itself can also lead to cell death.  Importantly, ischemic preconditioning (IPC) has been shown to limit the damaged induced by ischemia/reperfusion and act as a cardioprotectant.  However, IPC is somewhat impractical in a clinical setting in terms of feasibility.  This study was able to show that a preconditioned non-ischemic remote trauma was also able to induce cardioprotection through neurogenic mechanisms.  Specifically they were able to show that stimulation of pain receptors located in the skin activated an antidromic spinal reflex, which in turn activated cardiac sympathetic nerves via calcitonin gene-related peptide (CGRP).  CGRP then induced NE and Bradykinin release, stimulating beta-AR and BK2R.  Activation of these receptors then caused an upregulation of PKC-epsilon (mediator of cardioprotection) and a down regulation of PKC-delta (mediator of cell-death via necrosis).  They believe that one of the cardioprotective effects of PKC-epsilon is the activation of mitochondrial Katp channels, which when inhibited attenuated the cardioprotective effects.  Conclusively, this study may provide to be extremely important clinically due to the ease and efficiency of the remote preconditioning of trauma on reducing I/R injury following an MI.

~JI 

Temporal Changes in the T1 and T2 Relaxation Rates (delta R1 and delta R2) in the Rat Brain Are Consistent With the Tissue-Clearance Rates of Elemental Manganese

Chuang, Kai‐Hsiang, Alan P. Koretsky, and Christopher H. Sotak. "Temporal changes in the T1 and T2 relaxation rates (ΔR1 and ΔR2) in the rat brain are consistent with the tissue‐clearance rates of elemental manganese." Magnetic Resonance in Medicine 61.6 (2009): 1528-1532.

One of the main points of recent MeMRI study was to determine the time course of influx and efflux of manganese in the RVLM following systemic injections of manganese.  As a trace mineral, manganese can be found in multiple forms in the body that effect both its rate of efflux from a cell and its effect on proton relaxation rates.  While we discuss signal intensity in our study as manganese uptake within a cell, signal intensity may also be dictated by the relative state in which the manganese is able to change the proton relaxation rates.  To determine the extent that changes in relaxivity (how effectively manganese is able to change relaxation rates) are contributing to changes in signal intensity MeMRI studies were done in combination with NMR, directly examining manganese concentrations in neuronal tissue.  Initial results showed that T1 and T2 relaxation times, as well as delta R1 and delta R2 peak 1 day after administrations of manganese, which corresponded to greatest concentrations of manganese in neuronal tissue.  In all cases manganese uptake was seen to be greater in the olfactory bulb than in the cortex.  Both relaxation rates and relaxivity levels return to baseline levels 4 and 5 weeks post injections, conclusive with absolutely manganese concentrations also returning to baseline at 4 weeks.  Conclusively, this study was able to show that T1 and T2 relaxation times were mainly influenced by transport of manganese in and out of the cell, compared to manganese taking different forms while remaining in the cell (relaxivity).  This is important, because it further validates manganese enhancement being measured following systemic injections of manganese is an appropriate representation of in vivo neuronal activity.

~JI

Voltage-dependent calcium currents in bulbospinal neurons of neonatal rat rostral ventrolateral medulla: modulation by alpha-2-adrenergic receptors

Li, Yu-Wen, Patrice G. Guyenet, and Douglas A. Bayliss. "Voltage-dependent calcium currents in bulbospinal neurons of neonatal rat rostral ventrolateral medulla: modulation by α2-adrenergic receptors." Journal of neurophysiology79.2 (1998): 583-594.

Previous research has shown that both high voltage activated (HVA) and low voltage activated (LVA) calcium channels are present in the brain and may play different physiological roles.  The HVA calcium channels consist mostly of N type, P/Q-type, and to a lesser extent L-type components which also contribute to different physiological processes such as neurotransmission, and calcium mediated gene expression.  Until this study, little was known about calcium channel properties in the RVLM.  However, it was known that C1 neurons in the RVLM expressed alpha 2A-adrenergic receptors that had been shown to inhibit calcium channels in other neurons via activation of an inwardly rectifying potassium conductance.  Through the combination of using 2A-adrenergic receptor agonists and antagonists with electrophysiology, this study was able to characterize calcium channels and their regulation via norepinephrine for the first time.  Results showed that the HVA current consisted mainly of N-type calcium channels, with a significant but lesser contribution from P/Q-type. They also found small, but present L-type calcium channel activity.  As for LVA current, they found it was present in the RVLM and was activated with a much lower (~-50mV compared to ~0mv) as expected.  Conclusively, the study revealed that norepinephrine mediated activation of alpha 2-adrenergic receptors is able to inhibit HVA currents, specifically N-type and P/Q-type, but not LVA currents.  This study was relevant to MeMRI studies because manganese enters neurons through active L-type calcium channels.  If little L-type calcium channel current is present, it would need to be addressed for future studies using MeMRI to examine neuroplasticity in the RVLM.

~JI

Rostral ventrolateral medullary but not medullary lateral tegmental field neurons mediate sympatho-sympathetic reflexes in cats

Barman, Susan M., and Hakan S. Orer. "Rostral ventrolateral medullary but not medullary lateral tegmental field neurons mediate sympatho-sympathetic reflexes in cats." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 299.5 (2010): R1269-R1278. Previous to this study, work by Dr. Barman and her laboratory had shown that the medullary lateral tegmental field (LTF) neurons set the pattern for resting sympathetic nerve activity (SNA), not the NTS, CVLM, or RVLM. She was very open in stating that sympathetic tone to most of the vasculature was not driven by the RVLM, and that the RVLM, CVLM, and NTS were not the only neuronal pathways that contributed to SNA modulation resulting from the baroreflex, chemoreflex, or vagal afferents. However, these hypotheses were based upon various microinjecitons into directly into the LTF while recording SNA, so this study looked to further characterize the role the LTF plays in the sympatho-sympathetic reflex in response to afferent stimulation. After the separate microinjection of 1) non-NMDA receptor antagonist (NBQX) 2) NMDA receptor antagonist (D-AP5) 3) muscimol into the LTF there were no differences seen in sympathoexcitatory response recorded from the right inferior cardiac nerve, following stimulation of either the left inferior cardiac afferent nerve (CN) or sympathetic afferent nerve (SN). This same protocol was then repeated in the RVLM and injections of NBQX reduced the sympatho-sympathetic excitatory response to ~25% from baseline levels, as well as increased the onset latency following the electrical stimulation to the CN and SN. Like in the LTF, microinjections of D-AP5 produced no significant differences in the sympatho-sympathetic excitatory response. Muscimol, however, like NBQX significantly reduced the responses to CN and SN stimulation. In most cases the response seen was negligible, with little to no excitation. Conclusively, there were no changes in the excitatory responses following identical microinjections into the NTS and CVLM. Results from this study disproved the original hypothesis and instead showed that the RVLM is a critical region involved in sympatho-sympathetic cardiovascular responses. Dr. Barman also goes on to hypothesize that non-NMDA receptors play a primary role in this reflex compared to NMDA receptors. ~JI

Efficacy of an L- and N-type calcium channel blocker in hypertensive patients with neurovascular compression of the rostral ventrolateral medulla

Aota, Yasuko, et al. "Efficacy of an L-and N-type calcium channel blocker in hypertensive patients with neurovascular compression of the rostral ventrolateral medulla." Hypertension Research 32.8 (2009): 700-705. It has been previously shown in clinical studies that essential hypertension may be linked to neurovascular compression (NVC), and that NVC maybe be mediating hypertension through increased levels of sympathetic nerve activity. Neurovascular decompression studies have been shown to decrease blood pressure in patient with essential hypertension, but requires a rather intricate invasive surgery. The purpose of this study was to examine the effects of non-invasive, sympatholytic, L- and N-type calcium channel antagonist (Cilnidipine) for the use of a treatment for essential hypertension mediated by NVC. In this trial, 46 patient with essential hypertension (22 –NVC, 24 +NVC) were treated with Cilnidipine for 16 weeks, with clinical follow ups at 0, 8, and 16 weeks. At 8 and 16 weeks, systolic and diastolic blood pressure in both groups was significantly reduced from baseline levels. Correspondingly, systolic and diastolic pressures were significantly reduced in the +NVC group, compared to the –NVC group. To examine SNA, norepinephrine levels were taken at each time point, revealing that baseline levels of norephinephrine were significantly higher in the +NVC group than the –NVCs. That being said, at 16 there was a significant decrease in NE levels in the +NVC, but not –NVC group. With no difference in NE levels found between the groups. Finally, after measuring left ventricular mass index (LVMI) at each time point, it was observed that LVMI decreased in the +NVC, but not the –NVCs. Conclusively, this study was able to show the use of Cilnidipine by patients with EH mediated by NVC may be a non-invasive antihypertensive treatment. However, because Cilnidipine is not only an N-type calcium channel antagonist, but also an L-type vascular calcium channel antagonist more studies are being done to distinguish if the results seen were due to sympatholic or vascular effects. ~JI

Role of voltage-gated L-type calcium channel isoforms for brain function

Striessnig, J., et al. "Role of voltage-gated L-type Ca2+ channel isoforms for brain function." Biochemical Society Transactions 34.Pt 5 (2006): 903-909. This is a more detailed study examining the role of both L-type calcium channel (LTCC) isoforms 1.2 and 1.3 within the brain. Like discussed previously in my last blog, because the LTCC agonist and antagonist do not show specific binding to either 1.2 or 1.3, it has been difficult to differentiate the roles each plays. However, more recently discovered, two groups of genetically modified mice have enabled scientist to conduct much needed studies better characterizing these two calcium channels. The first mouse group Cav1.3-/- has a selective knockout for all Cav1.3 channels. Whereas the second modified mouse group Cav1.2-/- has a modified α1 subunit that inhibits the binding of agonists and antagonists from the channel. With the creation of these mice, creative studies can now selectively examine either isoform 1.2 or 1.3 depending on which pharmacological substances are utilized. Recent studies have now found that homologous knockouts of isoform 1.3 causes complete deafness, as well as sinoatrial node dysfunction. These results lend to the hypothesis that 1.3 specific calcium channels play a vital role in cochlear hair and sinoatrial node cell signaling transduction. Similarly, experiments utilizing Cav1.2-/- have revealed the importance in Ca2+ influx through Cav1.2 in regulating smooth muscle and cardiac contractility. This study also reiterates the importance of both isoforms in synaptic plasticity pertaining to memory, learning, and the storage of fear induced memories. Interestingly, when sensitivity to direct Ca2+ channel activators was inhibited in Cav1.2-/-, the efflux of glutamate from neurons in the ventral striatum was completely abolished, providing evidence for the importance of Cav1.2 in neurotransmission of glutamate. Again, I think this paper provides plausible evidence that L-type calcium channels may play an important role in synaptic plasticity within the RVLM. ~JI

The role of L-type voltage-gated calcium channels Cav1.2 and Cav1.3 in normal and pathological brain functions

Berger, Stefan M., and Dusan Bartsch. "The role of L-type voltage-gated calcium channels Cav1. 2 and Cav1. 3 in normal and pathological brain function." Cell and tissue research 357.2 (2014): 463-476. There are multiple types of voltage-gated calcium channels (VGCCs) including L-type, T-type, P/Q-type, R-type, and N-type that are defined by their pharmacological responses. Each channel is made up of five subunits, with the main pore forming subunit being alpha1. For this discussion I will mainly be focusing on the L-type calcium channels(1.1-1.4) which are believed to be the channels that allow manganese entry into a depolarized cell when using manganese enhanced MRI. More specifically, I will be focusing on 1.2 and 1.3 which are expressed by in large in the body compared to the more restricted 1.1 and 1.4. Cav1.2 and Cav1.3 can be found on multiple organs in the periphery including the hear, smooth muscle, pancreases, and adrenal glands, but most importantly for my studies, both of these channels are also expressed on neurons in the brain. With some cells expressing both 1.2 and 1.3, leading to the hypothesis that each channel may have its own specific function and importance. Some studies using radioreceptor assays have suggested that 89% of all Cav isoforms in the brain are 1.2, where as only 11% are 1.3. However, other studies utilizing western blotting have stated that within neurons in the hippocampus, cerebral cortex, and cerebellum 1.2 and 1.3 isoforms are equally abundant. As for location within a cell, it appears 1.2 are mostly found on the post synaptic dendrites, compared to the 1.3 which is most dense around the cell body. Electrophysiologically, 1.3 isoform channels are activated more rapidly and in more hyperpolarized membranes. They are also inactivated with a slower current than 1.2 isoforms. In both channels calmodulin acts as an imperative calcium sensor that 1) initiates the inactivation of the calcium channel preventing intracellular calcium toxicity 2) causes phosphorylation of the channel which increases the probability of an open state during repeated or prolonged activation and 3) enables the expression of calcium dependent genes within the cell. Unfortunately, there still is not a well established antibody for the isoform 1.3 so these studies may be less reliable then more recent studies being undertaken using Cre recombinase knockout mice. Using genetically modified mice, pharmacological experiments have showed that LTCCs play a role in synaptic plasticity involving learning and memory. Interestingly, injections of LTCC antagonists into the hippocampus have shown increases in acquisition and retention of spatial reference and working memory. Correspondingly, chronic injections into older animals has shown prevention of age-related hippocampal-dependent memory loss. It is believed this change is linked to the loss of NMDA-receptor-independent form of late long-term potentiation. Related, it has also been shown using LTCC antagonist injections into the amygdala that blocking LTCC also blocks the formation of fear memories. Finally, other studies have shown a link between LTCC's and the modulation of the mesoccumbal dopamine signaling pathway, which plays a major role in the reward system and addiction. Compared to animal studies, few human studies have been done analyzing LTCCs. However, in the past few years the hypothesis that LTCC's play an important role in psychiatric diseases is becoming more and more accepted. Stemming from patients with Timothy Syndrome, upregulated LTCC activation leads to the upregulation of tyrosine hydroxylase expression, causing increased concentrations of norepinephrine and dopamine. They have also shown that stimulation of TS-mutated calcium channels 1.2 cells led to dendritic retraction. Since the GWA the CACNA1C gene, associated with the alpha1 subunit on Cav1.2, was identified as a common risk factor allele for bipolar disorder, schizophrenia, and major depression. As for physiological defects in the isoform 1.3, recent studies are showing Cav1.3-mediated vulnerability of the dopaminergic neurons affected by Parkinson's disease. Specifically, increases in calcium entry increases alpha-synuclein aggregates present in Parkinson's disease. Conclusively, very few studies have been done examining the role of VLCC's in normal human cognition up to date. I think studies examining the role of L-type calcium channel activity within the RVLM would be interesting, and may shed light on differences in neuroplasticity between sedentary and physically active animals. ~JI

Magnetic Resonance Imaging of Cortical Connectivity

Canals, S., et al. "Magnetic resonance imaging of cortical connectivity< i> in vivo." Neuroimage 40.2 (2008): 458-472. Understanding the vast potential of manganese-enhanced MRI as a neuronal tract tracing tool for examining complex neural connections, this study by Canals et. al looked to characterize direct injections of manganese into the cerebral cortex, as well as better define efferent connectivity from of the somatosensory and motor regions. Specifically, a variety of manganese concentrations (0.05-0.80M) and volumes (10-200nl) were injected into the somatosensory and motor cortex using an osmotic pump and cannulae. Following cranial injections (multiple protocols), immunohistochemistry and fluorescence microscopy were performed to determine the extent of neuronal damage/toxicity. An initial study using injections of 200nl .4M and .8M MnCl2 in water revealed that injections of 200nl .8M MnCl2 produced extensive cell death and astrogliosis. This is important because cell viability is critical when performing longitudinal studies. Also, damaged tissue compromises the spread and uptake of acute manganese, effecting acute studies as well. A second protocol using multiple injections with varying manganese concentrations with an array of pH's demonstrated that injections of higher concentrated manganese and greater acidity produced larger neuronal lesions and increased astrogliosis. On the basis of the characterization results, this study proclaimed infusion of 100mM MnCl2 in 80nl at a rate of .5nl/min produced the most optimal results with the lowest toxicity. The later half of the study was then designed to investigate the efferent projections from the somatosensory and motor cortex using the previously defined manganese solution against non-optimal solutions (acidic, hypertonic,and concentrated). Importantly, the results from this second study showed better signal enhancement following an injection of 8nmol 100mM MnCl2 pH 7.3 than 8nmol 800mM MnCl2 pH 5.5 in multiple brain regions the somatosensory cortex projects to, including the thalamus and S2. Also, using the optimal manganese solution also allowed for the visualization of trans-synaptic tracing. With an appropriate manganese solution identified, they then examined the possibility of enhancing multiple neuronal pathways with multiple injection sites. Conclusively, it was determined using 3D analysis that it is possible to not only image multiple neuronal pathways, but also to image and analyze the overlap between these pathways using statistical connectivity maps. Interestingly, they were also able to study and quantify connectivity strength though the corpus callosum using this same 3D statistical mapping analysis. Conclusively, the final study revealed that using a microosmotic pump to slowly infuse large amounts of manganese (24nmol .25ul/hr compared to the 8nmol .5nl/min)allowed for visualization of brain regions previously unidentified using the initial faster injection. However, there are still experiments being done to mearue teh time course of transport and effective injection site viability. Overall, this was a large descriptive study that examined many factors of direct injections of MnCl2 into the brain, that will be an extremely helpful reference for our future studies. ~JI

Overexpression of angiotensin-converting enzyme 2 attenuates tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats

Wang, Yang-Kai, et al. "Overexpression of angiotensin-converting enzyme 2 attenuates tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats." American Journal of Physiology-Heart and Circulatory Physiology (2014). This article was focused on the role of ACE2 in the RVLM and its effects on hypertension. Overexpression of ACE2 was achieved using microinjections of lentivirus, containing a GFP tag expressed upon transcription, into the RVLM. WKY, SHR, SHR-leniGFP, and SHR-lentiACE2 were observed for 6 weeks and measured for BP, HR, Norepinephrine excretion, and glutamate concentrations within the RVLM. Acute microinjection studies were also done with Kyn to look at responses to glutamatergic inhibition in the RVLM. It was found that three weeks following injections, BP and levels of NE excreation were significantly decreased from baseline in lentivirus-ACE2 positive animals but not in lenti-shams, SHR, or WKYs. It was also observed after acute microinjecitons of Kyn into the RVLM that SHR-ACE2 animals had decreased RSNA activity, and an attenuated decrease in blood pressure compared to the SHR-GFP animals. Lastly, it was discovered that with an upregulation of ACE2/Ang(1-7) there was a corresponding downregulation of the AT1R, NMDA receptor 1 protein, and GluR5/6/7 proteins. Contrarily, there was an upregulation of Mas receptor that Ang(1-7) is known to innervate. Based on this evidence the authors concluded that ACE2 attenuates tonically active glutamatergic inputs to the RVLm in SHR. They discussed possible mechanisms being through the upregulation of Mas interfering with glutamatergic neurotranmission at the level of the pre-synaptic cleft. However, further research is needed in order to characterize the exact mechanism in which ACE2 is attenuating glutamatergic input. Conclusively, this study provides evidence for the possible use of ACE2 as a therapeutic agent for hypertension. ~JI

Effects of exercise training on dendritic morphology in the cardiorespiratory and locomotor centers of the mature rat brain

Nelson, Amanda J., et al. "Effects of exercise training on dendritic morphology in the cardiorespiratory and locomotor centers of the mature rat brain." Journal of Applied Physiology 108.6 (2010): 1582-1590. This study was very similar to Nick's structural study in that it examined morphological plasticity of neurons involved with cardiac sympathetic modulation. However, this study looked at respiratory and locomotor brain regions as well. Another distinguishable difference between the two studies was the age at which the animals began exercising. This study was interested in examining neural plasticity in adult rats unrelated to development. To do this they started exercise training the rats only after the animals were 91 days old, and maintained spontaneous free-running for 50 days following initiation. The reason I was interested in this paper particularly was because we are having trouble imaging the smaller rats (~99g), and in order to do a longitudinal study it is imperative we collect reliable data at each time point. I thought this may be helpful because we have not looked at plasticity in rats older than ~18 weeks of age 11-13wks running. These rats were 13 weeks old when the exercise training began and ~20 weeks old when the sholl analysis was done looking at dendritic arborization. Unfortunately, no differences were found between exercising and sedentary animals dendritic branching within the RVLM at 20 weeks of age. Based on these results I would avoid prolonging my longitudinal study looking at neuronal activity plasticity to an older subset of rats if at all possible, which I believe is. They did, however,see structural plasticity in other brain regions at this stage in life, including in the NTS, posterior hypothalamus, periaqueductal gray, and the cuneiform nucleus. In the majority of these brain regions though, they found that the effect size was not as great in older rats as it was in the younger rats. One of the major limitations to this study was that they were not able to differentiate if the decreasing plasticity with age was due to increases in age, or decreases in running because older rats naturally run less than their younger counter parts. I wondered if they looked at a correlation between individual animals running and plasticity because this show evidence for one possible versus the other. ~JI

Running Throughout Middle-Age Improves Memory Function, Hippocampal Neurogenesis, and BDNF Levels in Female C57BI/6J Mice

Marlatt, Michael W., et al. "Running throughout middle‐age improves memory function, hippocampal neurogenesis, and BDNF levels in female C57BL/6J mice." Developmental neurobiology 72.6 (2012): 943-952. I found this article to be relevant because I am interested in comparing BDNF levels within the RVLM of sedentary vs physically active animals. I am also interested in reading more about effects of exercise later in life than the "adolescent phase" due to the great technical difficulties we are facing imaging younger rats for MeMRI. Specifically, this article discusses the beneficial effects seen from chronic running during later stages in life. The model they used was a nine month old mouse model that exercised freely for 1 month prior to the first set of experiments and then another 5 months prior to the second set of experiments. As stated, at both 1 month and 6 months of exercising anxiety, memory, and motor tests were performed. Then at 8 months post exercise each of the animals were sacrificed and BDNF levels and BrDu positive neurons were examined. They found that after one month of exercising, mice exhibited less anxiety with increased central area during an arena test, as well as increased distance traveled. They also exhibited a tend toward increased latency between falls in a rotarod performance test measuring motor abilities. It was then seen that after 6 months of exercise animals showed increased social memory by increasing the amount of time within the target quadrant in a Morris water maze. These animals also trended towards a decreased number of falls during the rotarod test. To support these findings animals that had chronically run had increased levels of mature BDNF peptide within the hippocampus after 8 months. Exercising animals also had a greater number of BrDu labeled cells in the dentate gyrus, along with an increase in the total number of new neurons. Concluding, this study was able to show that chronic running later in life (9 months) improved spacial memory, motor skills, as well as decreased anxiety. This may be due to increased neurogenesis and neurotrophins in the hippocampus after 8 months of free exercise. I think it would be informative to compare BDNF levels, as well as neurogenesis within the RVLM between sedentary and physically active population. ~JI

The entry of manganese ions into the brain is accelerated by the activation of N-methyl-D-aspartate receptors

Itoh, K., et al. "The entry of manganese ions into the brain is accelerated by the activation of< i> N-methyl-d-aspartate receptors." Neuroscience 154.2 (2008): 732-740. There are many advantages of using MeMRI to study in vivo neuronal activity. However, there is the disadvantage of having to anesthetize the animal during the imaging which has an effect on overall brain functioning. Previous studies done by Lin/Koretsky and Aoki have demonstrated that manganese enhancement in MR images is dampened the deeper an animal is in anesthesia. That being said, no studies had been done looking at effects of manganese enhancement across different methods of anesthesia until Itoh in this study in 2008. They found that most anesthetics including isoflurane, urethane, and pentabarbitol did not have significant affects on manganese enhancement. Where as the use of ketamine significantly decreased manganese enhanced contrast. Unlike the other anesthetic that act by potentiating GABAergic pathways, ketamine acts as a partial antagonist for NMDA receptors. An NMDAR under natural conditions is acted upon by glutamate to then activate glutamatergic neurons via the influx of calcium. The observation of decreased manganese enhancement in the presence of ketamine then led to further questions about the entry of systemic manganese into the cerebral spinal fluid and eventually brain tissue. Using various NMDA, GABAa, and AMPA agonists and antagonists this study found that NMDAR mediated glutamatergic excitation plays a major role in influencing manganese enhancement in MRI. Both NMDAR antagonist significantly decreased manganese enhancement within the ventricles (other brain regions did not show differences because images were only taken up to three hours post MnCl2 injections). Oppositely, NMDAR agonist produced significantly increased manganese enhancement compared to controls. AMPA antagonist did not appear to change manganese enhancement, while GABAa antagonist also produced increased manganese enhancement. A limitation to this study is that they did not address effects resulting from changes in the NMDARs within brain tissue itself, which is important to our studies looking at plasticity in neuronal activity in the RVLM of sedentary and physically active rats. I would hypothesized by extrapolating from this study however, that sinoaortic dennervated rats will show an increase in manganese enhancement within the RVLM when compared to the non-dennervated control animals. ~JI

Testing the calcium hypothesis of aging in the rat hippocampus in vivo using manganese-enhanced MRI

Bissig, David, and Bruce A. Berkowitz. "Testing the calcium hypothesis of aging in the rat hippocampus in vivo using manganese-enhanced MRI." Neurobiology of aging 35 (2014) 1453-1458 This study used MeMRI to examine how aging affects changes in L-type calcium channels in the hippocampus. It has been hypothesized that increases in calcium influx through L-type calcium in the hippocampus that is associated with cognitive decreases. By utilizing MeMRI Dr. Berkowitz was able to non-invasively characterize L-type calcium channels in a longitudinal study. They found that aged rats have significantly increased manganese uptake in pyramidal neurons in the hippocampus compared to their younger selves. Although they were not able to look at mechanistic changes, the trends that they seen in increases in manganese uptake are consistent with the hypothesis of hippocampal plasticity linked to cognitive decline. In relation to our study this paper talks about imaging control rats as well. They found that their were no differences in the older control rats regions of interest when compared to the younger rats regions of interest. They also looked at differences in manganese uptake in animals that had been previously injected versus non-injected animals. There were no differences in animals that had been previously injected versus not injected. In the discussion they briefly talked about changes in the BBB with age that may affect manganese uptake over time. This is important to what we study since we are doing longitudinal studies. ~JI

Age-Related Impairment in Choroidal Blood Flow Compensation for Arterial Blood Pressure Fluctuation in Pigeons

Reiner, Anton, et al. "Age-related impairment in choroidal blood flow compensation for arterial blood pressure fluctuation in pigeons." Investigative ophthalmology & visual science 52.10 (2011): 7238-7247. It is well known in our area of research that increased sympathetic nerve activity is a risk factor for many cardiovascular disease states. Well we mainly focus on peripheral vascular consequences associated with cardiovascular disease states, there is also end organ damage that can have other damaging affects. This study in particular is examining changes in the regulation of choroidal blood flow (ChBF) with age. Similarly to the kidneys, the choroid will regulate blood flow by changing vascular resistance in correlation with blood pressure. In healthy individuals, an increase in blood pressure will cause an increase in vascular resistance and the maintenance of blood flow. Vice versa, if blood pressure decreases the choroid will vasodialate as much as possible to compensate and maintain blood flow. Using Doppler flowmetry to measure ChBF and telemetry in pigeons ranging from .5 to 17 years, Fitzgerald's laboratory was able to longitudinally charaterize ChBF regulation with age. They found that between a arterial blood pressures (ABP) of approximately 55mmHg-135mmHg pigeons younger than 8 years of age were able to properly maintain ChBF with changes in vascular resistance. However, in pigeons older than 8 years of age this baroreflex was impaired. Specifically, above 90mmHg and below 60mmHg these pigeons had a positive correlation between changes in ABP and changes in ChBF. It was also found that between 60mmHg-90mmHg older pigeons were only able to maintain ChBF between 60 and 70% of basal level. Where as, in young pigeons ChBF was maintained at 100% between 55mmHg-135mmHg. To support both sets of data, a positive correlation between changes in vascular resistance and ABP existed in the younger cohort of pigeons, but not the older cohort. These data suggest that regulation in ChBF is effected with age, and Reiner suggest two possible mechanisms. Either changes in neurogenic baroreflex regulation or myogenic regulation at the level of the vasculature. However, more studies need to be done in order to determine which and or if both are playing a role. Conclusively, the study determined this loss of ChBF regulation may in part be responsible for onset ocular disease states, and that these disease states may be perpetuated by risk factors that alter sympathetic nerve activity such as hypertension or age. ~JI

Manganese-enhanced magnetic resonance imaging reveals increased DOI-induced brain activity in a mouse model of schizophrenia

Malkova, Natalia V., et al. "Manganese-enhanced magnetic resonance imaging reveals increased DOI-induced brain activity in a mouse model of schizophrenia." Proceedings of the National Academy of Sciences (2014): 201323287. It is well known that mental illnesses, such as schizophrenia, are difficult to study in an animal model for a number or reasons, so many laboratory do their best by studying mental illness like symptoms. For this study, in order to better understand the role that the environmental risk factor maternal immune activation (MIA) plays in schizophrenia and autism, Malkova and colleges examined DOI-induced brain activity. DOI, 2,5-dimethoxy-4-iodoamphetamine, is a drug that through activation of the serotonin receptor 5-HT2AR, produces similar hallucinations as experienced in schizophrenia. Multiple methods (quantitative PCR, behavioral analysis, and MeMRI) were then used to compare differences in DOI-induced activity in control versus MIA animals. They found that there was an upregulation of drug induced head twitches, manganese accumulation in multiple areas of the brain (primary and secondary motor cortex, caudate putamen, medial group of the dorsal thalamus, and parafascicular thalamic nucleus), and expression of 5-HT2A receptor and its down stream signaling molecules in MIA mice compared to controls. What I found most interesting in this paper was the MeMRI protocol. For this experiment they first took baseline scans one week prior to the injection of manganese. Following the the I.P injection a week later they then imaged the animals at 24hrs, injected the animals with DOI, and then imaged them again 27hrs post the I.P injection. For statistical analysis, they then compared baseline levels to the 24hr image as well as the 27hr image. This may be applicable for our studies looking at differences in exercising versus sedentary brain activity of the RVLM. If we were to take baseline scans, inject manganese one week later, image the animals at 24hrs, raise and/or lower blood pressure for 8 hours and then immediately image the animals at 32hrs we may see different results then previously observed. This is one possibility I had not previously thought of. ~JI

Non-invasive, in vivo monitoring of neuronal transport impairment in a mouse model of tauopathy using MEMRI

Bertrand, Anne, et al. "Non-invasive,< i> in vivo monitoring of neuronal transport impairment in a mouse model of tauopathy using MEMRI." NeuroImage 64 (2013): 693-702. The tau protein is a well define protein involved in the stability of microtubules, found mainly in the axonal compartment of neurons. Unfortunately, under certain disease states referred to as tauopathies, such as Alzheimer's disease, there is a dysfunction in the tau protein due to over expression or increased phosphorylation that leads to aggregated tau protein. This tau protein aggregation is thought to blunt microtubule stabilization found under normal conditions and cause impairment of axonal transport, further leading to synaptic dysfunction. Previous to this study, the only methods used to examine axonal transport were either invasive in vivo techniques or in vitro techniques that came with many limitations. Anne Bertrand et. al, were interested in developing manganese enhanced MRI (MEMRI) as a non-invasive, in vivo technique that would allow for the observation of axonal transport viability under normal and tauopathy conditions. To do this a cross section study was done with both transgenic (tau pathology) and control mice, each population were injected intranasally with 1.5ul of 5M manganese chloride and groups were imaged at 3, 6, and 9 months at 1, 4, 8, 12, 24, 36, 48hrs, and 7-10 days following the injection. Each cohort was then sacrificed and used for immunhistochemistry examining tau protein concentrations. What Bertrand found was that there were significant decreases in MnCl2 signal propagation between WT and transgenic mice at 6 months in age in two tissue layers throughout the olfactory track that were examined (glomerular and mitral cell layers). There was also a significant decreases in signal propagation due to age between the transgenic mice 3 months of age and 6 months of age within the glomerular and mitral cell layer (smaller differences seen in the mitral cell layer). After immunohistochemistry, they were then able to correlate tau pathology to the observed MEMRI parameters. In this case, there was a significant negative correlation between tau pathology and signal propagation of manganese. Correspondingly, there was a greater correlation to dendritic tau staining and the MEMRI parameters compared to somatic tau staining and MEMRI parameters. Interestingly, this paper also did GFAP staining for astrogliosis to rule out differences in manganese accumulation from discrepancies in astroglial cells. They found no differences between GFAP staining in the WT and transgenic mice. Conclusively, this study demonstrated that MEMRI can be used as a viable in vivo technique for the analysis of axonal transport function, which may possibly be used for clinical analysis of tau protein as a biomarker for many disease states or possibly treatment options. ~JI

Chronic absense of baroreceptor inputs prevents training-induced cardiovascular adjustments in normotensive and spontaneously hypertensive rats

Ceroni, Alexandre, et al. "Chronic absence of baroreceptor inputs prevents training‐induced cardiovascular adjustments in normotensive and spontaneously hypertensive rats." Experimental physiology 94.6 (2009): 630-640. It has been shown in both rats and humans, that a low intensity exercise regiment can be used to lower resting heart rate and consequentially blood pressure, in both normotensive and hypertensive individuals. Currently, cardiovascular responses during exercise are known to be driven by both a feedforward pathway (central command) and a feedback pathway (baroreceptors, chemoreceptors, muscle metaboreflex) that are synergistic in modifying cardiovascular responses. However, it is still unclear as to the underlying mechanisms during exercise drive the beneficial . Dr. Michelini's laboratory hypothesized that an intact baroreceptor reflex is necessary for training-induced adjustments of cardiovascular control to occur. To investigate their hypothesis they performed sino-aortic denervations in both normotensive (WKY) and spontaneously hypertensive rats (SHRs) and looked at the effects of low intensity exercising training in both sham WKYs and SHRs compared to SAD WKYs and SHRs. They found that a sino-aortic denervation completely blunted the affects of exercise training in both WKYs and SHRs compared to what was seen in both sham populations. More specifically, the SAD populations did not experience bradycardia or decreased blood pressure after a training as the sham groups (normotensive did not experience decreases in BP). They were however, able to observe reduce pressure variability and heart rate variability in SAD SHRs on an exercising regiment compared to the SAD SHRs not exercising. Overall, the study was able to show that the baroreceptor reflex input plays some role in allowing for cardiovascular adjustments driven by exercise training. ~JI

Asymmetrical changes in lumbar sympathetic nerve activity following stimulation of the sciatic nerve in rat

Korim, Willian Seiji, et al. "Asymmetrical changes in lumbar sympathetic nerve activity following stimulation of the sciatic nerve in rat." Brain research 1391 (2011): 60-70. The somatosympathetic reflex (SSR) can be observed as responses in post ganglionic sympathetic nerves after the stimulation of Type 2 and 3 sensory fibers. Before this study it was known that this reflex produced differential responses in ipsilateral and contralateral hindlimb blood flow (HFB) following activation of the sciatic nerve. However, how these changes were occurring was unknown, Pilowsky's laboratory hypothesized that changes in HBF was due to differential changes in lumbar sympathetic nerve activity (lSNA) and this was being driven by supraspinal structures (RVLM). To do this sciatic nerve stimulation was accomplished while both ipsilateral and contralateral lSNA was recorded under a variety of conditions (normal, cervical spinal transections, and injections of muscimol into the contralateral RVLM). Before the the cervical spinal transections and blockade of the RVLM it was observed that following sciatic nerve stimulation a differential response was observed at the level of the lSNA. Specifically, the ipsilateral recording showed inhibitory potentials corresponding to decreases in lSNA, while contralateral recordings showed sympathoexcitatory potentials corresponding to increased lSNA. This is consistent with previous literature showing increases in ipsilateral HBF and decreases in contralateral HBF following stimulation of type 2 and 3 sensory fibers. The second observation, was following cervical spinal transections both ipsilateral inhibitory and contralateral excitatory response were diminished. This suggests the reflex is controlled partially by a supraspinal structure. Pilowsky hypothesized that the RVLM was taking part in this reflex and initially did microinjections of muscimol into the contralateral RVLM. Following the microinjections all inhibitory and excitatory responses were abolished. To further investigate, microinjections of glutamate antagonist (kynurenic acid) were also injected into the RVLM and it was seen that only the sympathoexcitatory responses were abolished. In conclusion, the microinjections studies suggest that the RVLM plays a role in the sympathoexcitatory SSR by activating pre-ganglionic sympathetic nerves in which then activate the post-ganglionic sympathetic nerves (lSN) controlling blood flow at the level of the hindlimbs. However, the exact role the RVLM plays in sympathoinhibition is still not clear and needs further investigation. ~JI

Altered Inflammatory Response Is Associated With an Impaired Autonomic Input to the Bone Marrow in the Spontaneously Hypertensive Rat

Zubcevic, Jasenka, et al. "Altered Inflammatory Response Is Associated With an Impaired Autonomic Input to the Bone Marrow in the Spontaneously Hypertensive Rat." Hypertension (2013): HYPERTENSIONAHA-113. This study was interested in the relationship between the immune system and cardiovascular disease, which they examined through the comparison of spontaneous hypertensive rats (SHR) and wild type Wistar-Kyoto Rats (WKY). They hypothesized that SHR would exhibit autonomic and endothelial dysfunctions, including increased inflammatory responses. Differences in femoral sympathetic nerve activity (fSNA) (innervation of bone marrow [BM]), alpha2a/beta2-adrenergic receptor expression, BM norepinephrine, BM imflammatory cells (ICs), edothelial progenitor cells (EPCs), and in vivo activity of the hypothalamic paraventricular nucleus (PVN)using MeMRI and GFP-pseudorabies virus (PRV)retrograde tracing. It is known that sympathetic drive in rats peaks at approximately 8pm and drops to its lowest levels around 11am, so most factors were measured both during the day and at night to review any possible changes in circadian-related sympathetic drive. Following the studies it was found that fSNA was increased 80% at night in the SHR compared to the WKY. There was also increased BM Nor and BM ICs during both the day and night in SHRs compared to WKY. Contrarily, BM EPCs were seen to decrease in SHR compared to WKY both during the day and night. However, blood ICs and blood EPCs were not seen to change from day to night time levels in the SHRs. Based upon the fact that IC's are known to compromise vascular integrity and EPCs are known to repair vascular damage, these results are conclusive with the hypothesis made. In order to compare sympathetic to parasympathetic drive in these animals levels of acetylcholine transferase and acetylcholine esterase were also measured showing decreases in both enzymes in the SHR population compared to the WKYs, insinuating a disregulation of sympathetic to parasympathetic drive. To support this, levels of fSNA were found to be significantly higher (25%)in SHR compared to normotensive control rats. Finally, both MeMRI and PRV retrograde tracing both revealed increased levels of in vivo neuronal activity of the PVN in SHRs compared to WKYs. Conclusively, this study showed an improper circadian-related balance between sympathetic and parasympathetic drive to immune organs (BM) that may be playing a role in perpetuating neurogenic hypertension. ~JI

Abstract: Manganese Enhanced MRI Assay of Spinal Cord Functional Connectivity

By: Xiaowei Zhang, Naomi Santa Maria, Samuel Barnes, and Russell E. Jacobs These studies looked to develop manganese-enhanced MRI for the examination of longitudinal spinal cord injury therapies. Previous to MeMRI extensive histology was needed on spinal tissue in order to characterize different therapies, these techniques however prevented longitudinal follow-up. Utilizing manganese as a retrograde tracer, 2,000 nanoliters of 200 mM MnCl2 was injected through a burr hole in the right lamina of adult female mice. The entire spinal cord and caudal portion of the brain were then imaged for four 400 micron sagittal T1 weighted slices 30 min, 8, 24, 48 and 72 hours after the injections. Interestingly, they were able watch manganese travel up the spinal cord into the brain stem, which pertains to what I am looking to do in the future. They saw that manganese intensity increased in the caudal part of the brain at 72 hours which is 24 hours post the time we are currently imaging the RVLM after spinal cord injections. We will be looking to examine a more acute time course of manganese uptake following spinal injections over the last month, this includes imaging the RVLM every 8 hours after injections. At some point it would be ideal to mimic these studies and observe manganese transport up the cord. However, more preliminary studies must first be done to facilitate this. ~JI