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UCSF Hyperpolarized MR #8-2: Neurological (2019)
- 1. Integration of Hyperpolarized MR Into Biomedical Research: Neurological 08/23/19 Lydia Le Page Chaumeil lab BioE297 seminars
- 2. Why the brain? Alzheimer’s traumatic brain injury Parkinson’s schizophrenia multiple sclerosis ALS stroke
- 3. Outline Preclinical - Healthy brain: developing and adult - Disorders: - Inflammation - Neurodegeneration - Probes with future potential - Challenges Clinical - Healthy brain literature - Current clinical trials Future potential
- 4. Preclinical: Healthy brain: developing and adult Chen et al. Developmental Neuroscience, 2016 Pyruvate to lactate conversion higher at younger age, decreased linearly with increasing age – 8 normal mice Age (days) Rateofpyruvatetolactate conversion(KPL) Future potential – the brain in healthy aging Feasibility shown in rats, mice, monkeys, and brain slices Harris et al. Sci. Rep., 2016
- 5. Preclinical: Neurological disorders – inflammatory cells Lactate:pyruvateratio Control Following treatment with an NSAID Inflammatory response to toxin Sriram et al, Theranostics, 2018 Najac et al, Scientific Reports, 2016 Macrophages Myeloid-derived suppressor cells – inflammatory cells involved in tumor development Increased urea production from HP arginine in inflammatory cells Immune cells such as macrophage and microglia change their metabolism when they are exposed to toxins One type of response, classical activation of immune cells, involves an increased production of lactate
- 6. Preclinical: Neurological disorders – in vivo inflammation Le Page et al., NMR Biomed., 2019 Injection of toxin Ipsilateral voxel for spectral analyis Contralateral voxel for spectral analyis Baseline 3 days 7 days Microglia/macrophages Astrocytes Baseline 3 days 7 days
- 7. Preclinical: Neurological disorders – neuroinflammation in MS Multiple sclerosis (MS) Baseline Week 4 ControlMultiplesclerosis Guglielmetti et al., PNAS, 2017 Lactate:pyruvate ratios Upregulation of PDK1 in activated microglia/macrophages in corpus callosum in MS mice Control Week 4
- 8. Preclinical: Neurological disorders – neuroinflammation in TBI Traumatic brain injury (TBI) Lactate:pyruvate ratios Guglielmetti et al., Scientific Reports, 2017 Baseline 12h 24h 7d 28d ControlInjury Pyruvate Lactate Bicarbonate DeVience et al., Scientific Reports, 2017 Contralateral Ipsilateral Microglia depleted Contralateral Ipsilateral
- 9. Preclinical: Neurological disorders – cognitive deficits High-fat-diet induced cognitive deficits Whole brain Lactate/pyruvate Time spent escaping a pool of water was increased in HFD- fed animals (6mo feeding) Choi et al., Molecular Brain, 2018
- 10. Preclinical: Neurological disorders – acute liver failure Brain metabolism changes in acute liver failure Intracranial hypertension is a severe complication Also brain edema is seen (Lactate+alanine)/pyruvate Acute liver failure Chavarria et al., NMR Biomed, 2015 Control 6h 12h 6h 12h
- 11. Preclinical: Neurological disorders - memory Memory acquisition and retrieval Harris et al., eNeuro, 2019 Then administered DCA multiple times 30 min before training First looked at brain metabolism before and 30min after DCA administration Concluded DCA exposure during training impaired long-term memory
- 12. Preclinical: Neurological disorders – probes with future potential in the brain Dehydroascorbic acid -> vitamin C1 Acetoacetate2 C2 pyruvate3 Glucose4 Lactate5 Acetate6 Oxidative stress/redox status TCA cycle disturbance Reduced glucose metabolism Changes in neuronal energy requirements Astrocyte metabolism 1Qin et al, Scientific Rep., 2018 2Najac et al, Scientific Rep., 2019 3Park et al., NMR Biomed., 2013 4Mishkovsky, Scientific Rep., 2017 5Takado et al, ACS Chem. Neuro., 2018 6Mishkovsky, Cer. Blood Flow and Metab., 2012
- 13. Preclinical: Neurological disorders – challenges Josan et al, MRM, 2012 ~ high and low doses of isoflurane ~ discussion of vasodilation with higher doses Marjanska et al, NMR Biomed, 2018 ~ pentobarbital, isoflurane, α‐chloralose, and morphine Miller et al, opened BBB with mannitol in pigs Peeters et al, opened BBB with focused ultrasound in mice Hurd et al, comparison of pyruvate and ethyl pyruvate Takado et al, BBB opened with ultrasound, saw increased HP lactate to pyruvate conversion Dose of anesthesia Crossing the blood brain barrier Future: Cho et al. (ACS Chem Biol, 2019) – development of HP arginine probe applied in a restrained awake mouse Pre- mannitol Post- mannitol Pyruvate Lactate
- 14. Clinical – neurological disorder trials in progress Current information from clinicaltrials.gov: - Small traumatic brain injury trial, C1, C2 pyruvate (16 total participants) - Responsible party: Jae Mo Park - C1 and C2 pyruvate feasibility study (28 participants, healthy brain maps) – to be applied to cancer - Responsible party: Jae Mo Park - Brain imaging of healthy volunteers
- 15. Clinical – healthy brain Park et al., MRM, 2018; Autry et al., MRM, 2019; Mammoli et al, IEEE Trans. Med. Imaging, 2019 Grist, NeuroImage, 2019 Gray matter, numerous cell bodiesWhite matter, mainly myelinated axon tracts T1-w pyruvate lactate KPL bicarbonate
- 16. Future - potential - Discover the source of the HP signal – brain cell types? - Technology can fit with existing methods for treatment monitoring HP 13C MRS MRI 1H SPECTROSCOPY BLOOD SAMPLING COGNITIVE ASSESSMENT PET IMAGING BIOPSIES
- 17. References Manuscripts Chen et al., doi: 10.1159/000439271 Harris et al., doi: 10.1038/s41598-018-27747-w Sriram et al., doi: 10.7150/thno.24322 Najac et al., doi: 10.1038/srep31397 Le Page et al., doi: 10.1002/nbm.4164 Guglielmetti et al., doi: 10.1073/pnas.1613345114 DeVience et al., doi: 10.1038/s41598-017-01736-x Guglielmetti et al., doi: 10.1038/s41598-017-17758-4 Choi et al., doi: 10.1186/s13041-018-0415-2 Chavarria et al., doi: 10.1002/nbm.3226 Harris et al., doi: 10.1523/ENEURO.0389-18.2019 Qin et al., doi: 10.1038/s41598-018-26296-6 Najac et al., doi: 10.1038/s41598-019-39677-2 Park et al., doi: 10.1002/nbm.2935 Mishkovsky et al., doi: 10.1038/s41598-017-12086-z Takado et al., doi: 10.1021/acschemneuro.8b00066 Mishkovsky et al., doi: 10.1038/jcbfm.2012.136 Josan et al., doi: 10.1002/mrm.24532 Marjanska et al., doi: 10.1002/nbm.4012 Cho et al., doi: 10.1021/acschembio.8b01044 Hurd et al., doi: 10.1002/mrm.22364 Miller et al., doi: 10.1038/s41598-018-33363-5 Takado et al., doi: 10.1021/acschemneuro.8b00066 Figures Macrophage: Wikimedia commons: File:Hematopoiesis (human) diagram en.svg Mitochondria: http://remf.dartmouth.edu/imagesindex.html Neuron:"Anatomy and Physiology" by the US National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) Program Astrocyte: http://togotv.dbcls.jp/ja/pics.html Brain, slide 11: By Henry Vandyke Carter - Henry Gray (1918) Anatomy of the Human Body Bartleby.com: Gray's Anatomy, Plate 745, Public Domain, https://commons.wikimedia.org/w/index.php?curid=541571 Manuscripts Peeters et al., doi: 10.1021/acschemneuro.9b00085 Park et al., doi: 10.1002/mrm.27077 Autry et al., doi: 10.1002/mrm.27743 Mammoli et al., doi: 10.1109/TMI.2019.2926437 Grist et al., doi: 10.1016/J.NEUROIMAGE.2019.01.027
Editor's Notes
- I will be describing some of the work done so far on how hyperpolarized MR is being applied to neurological biomedical research, not including cancer.
- Why are we interested in the brain? There are many debilitating brain diseases that we don’t fully understand and therefore struggle to treat, such as those listed here. There is obviously much active research to understand these diseases Changes in metabolism are seen in disease progression, and so visualizing this both during progression and on treatment would be a valuable measurement, and may lead to mechanistic insights and new avenues of research, which might in themselves lead to new treatments
- Hyperpolarized MRS has the potential fill this existing gap, in, as we’ve heard in previous lectures, its ability to provide measures of in vivo metabolism. In this talk, I will present examples from the current literature to give an idea of the breadth of this developing field, both preclinical and clinical. Relevant work I will talk about includes that in the healthy brain, in some brain disorders, and then also consideration of the challenges of the technique. Clinically, it is early days, but I will cover a few papers and the status of clinical trials.
- Most of my discussion will be mentioning studies using HP pyruvate for this first section. It is always going to be tricky to understand the brain in disease when there is so much to understand still in the healthy brain. Metabolic changes, for example in glucose metabolism, occur throughout life. This paper from UCSF in 2016 imaged mice from post-natal day 18 with hyperpolarized pyruvate, showing decreasing pyruvate to lactate conversion with age. They stated that lactate production was needed more at a younger age to support, amongst other things, neuronal fuel requirements, and this fit with the observation from MRS. There is also potential here to study the healthy aging brain with hyperpolarized MRS, but I have not seen publications on this yet. Feasibility of HP imaging has been shown in rats, mice and monkeys – further one technique worth highlighting here is tissue slices in the MR system, which has recently been applied in the brain. Rat brain slices showed metabolism of HP pyruvate, so could be used to gain mechanistic insights in a sensitive system from disease models.
- Moving into disorders in the brain. One factor which is increasingly appreciated to play a role in several brain diseases such as Alzheimer’s and multiple sclerosis is inflammation. Inflammation has also been investigated in other fields such as liver inflammation, arthritis, and myocardial infarction but we're now starting to apply it in neurological disorders. Immune cells such as macrophages and microglia, the resident immune cells in the brain, alter their metabolism when they are activated, such as when responding to a toxin. One type of response is the so-called classical activation, which involves an increased production of lactate. This was exploited in Renuka’s work in Theranostics which used a macrophage inflammatory cell line to show the value of hyperpolarized MR – it was possible to detect the increased production of lactate after classical activation of the cells by a toxin – lipopolysaccharide. Subsequent treatment with an NSAID was also detectable by MRS. Chloe Najac’s work with HP arginine exploited a characteristic of specific type of inflammatory cells – those involved in tumor development She showed that the HP arginine probe could be used to distinguish between the inflammatory cells and control bone marrow cells, based on the levels of the relevant enzyme arginase and conversion of arginine to urea.
- Moving in vivo! From our lab – we injected the same toxin as Renuka used in cells into one side of the mouse brain and used HP pyruvate to see if we could detect the inflammatory response. Here you see the injection site and the heat map of lactate, with red indicating higher signal. This was seen alongside increased staining for immune cells microglia, macrophages and astrocytes.
- To help us understand better the direct links between brain inflammation and the HP signal, we can look at further work from our group. This paper led by Caroline Guglielmetti applied HP MRS to a mouse model of multiple sclerosis, again showing increased pyruvate to lactate conversion in the disease model. Activated microglia and macrophages were seen, alongside increased PDK1, which inhibits oxidative metabolism via pyruvate dehydrogenase. This study is cool because then, an MS model with a specific genetic background was generated which meant that microglia/astrocytic activation was inhibited – and in this case, the increased pyruvate to lactate signal was no longer observed, giving strong support to a causal link.
- Traumatic brain injury is another neurological disorder with many unanswered questions; a limited understanding of the processes occurring, and no real treatments available. Two preclinical studies thus far have begun to apply HP MRS, using mouse models involving cortical impact. Returning to the inflammation aspect, a study from our lab first showed lactate:pyruvate ratios increasing after injury. Then, to investigate the inflammatory links, a drug was given to deplete the microglia…the increased lactate was no longer observed! An important second study by DeVience et al also showed increased lactate, adding decreased bicarbonate, from HP pyruvate following impact. A strength of imaging really shines through in these studies – the ability to image longitudinally provides very valuable datasets.
- There are a few other initial studies into interesting aspects of brain metabolism in disease, which really confirm the broad possibilities for HP applications. The change in lactate to pyruvate ratio (or lactate+alanine in one of the studies) is still the output from these works – in a high-fat-diet model with cognitive deficits, a liver failure model with effects on the brain, and in a study of memory Starting with this first study, the authors used a high-fat-diet that caused cognitive deficits. These animals showed an increase in time to find the escape platform in a pool of water following training – this is a measure of memory. I’ve put a typical display here – the lines are the paths of the mouse. This was then coupled with observation of increased lactate/pyruvate ratios in the brain!
- The second study here used a model of acute liver failure where intracranial hypertension is a severe complication. These authors could distinguish between the liver failure model and the controls, when they used HP MRS to look in the brain They saw increased (lactate+alanine) 12h after induction of acute liver failure, compared to no increase in control animals.
- This last in vivo preclinical study by Harris investigated memory and metabolism. They treated animals with dichloroacetic acid (DCA), stimulating oxidative glucose metabolism. They imaged before and 30 minutes after DCA treatment, and saw this decreased lactate production. They then administered DCA multiple times, always 30 minutes before training - the same training as in the first study, the paltform in the pool of water - and looked at how long the mice took to find the platform. The DCA animals took longer and they concluded that DCA exposure during training impaired long-term memory. The first and last studies here show especially interesting integration into neurology, given their performing of HP MRS alongside behavioral studies: widely used in the neurology community.
- As we’ve learnt previously, there are many challenges to overcome when it comes to developing novel HP probes, which is a key target if the technology is to reach its full potential. Listed here are some probes in the literature that have been developed and hold potential for assessing neurological changes. It would be super to be able to image these targets of oxidative stress, TCA cycle disturbance, reduced glucose metabolism and changes in specific brain cell energy requirements, such as neurons and astrocytes. Hopefully some of these probes can help us achieve that goal. Some of these are further along than others, for example DHA, which was recently shown to provide viable data in the rat brain, with the readout responding to treatment.
- Further significant challenges exist which should definitely be taken into consideration when planning future neurological studies. I will mention two of the great challenges here: The first is the dose of anesthesia – widely used for the majority of animal studies but which will have an effect on metabolism. Not limited to the brain, this issue has been investigated previously albeit no particular solution is available just yet. Isoflurane has a vasodilation effect, discussed in this first paper, enabling more pyruvate entry. Further in Marjanska’s work pyruvate to bicarbonate conversion was seen to be greatest when using morphine of these anesthetics. One interesting progression was seen in this recent paper by Cho and colleagues who performed studies with no anesthesia, only restraining the animal – this would be a very interesting method to implement widely, but does require training before imaging. We would also need to ensure that the stress response is negligible and not affecting our data. For the brain, one of the other biggest issues is ensuring that the HP probes cross the blood brain barrier. The blood brain barrier is a cell border between the external vasculature and the brain, which prevents, for example, pathogens getting into the brain. It does also limit the passage of drugs, and our probes. A few studies have demonstrated this limitation, shown here, and this must be taken into consideration when analyzing data or developing new probes. This image is from Jack Miller's paper where they show lactate production in the pig brain only after opening the blood brain barrier with mannitol. The other papers here explore a more lipophilic alternative to pyruvate, and opening the blood brain barrier with ultrasound. Further, it is important to establish whether changes in BBB permeability occur due to treatment or disease, as this can affect the interpretation of data.
- Despite the recency of HP work moving into the neurological field, there are already clinical trials recruiting and ongoing, alongside obviously the multiple cancer trials. I'd like to highlight two at UT Southwestern (Dallas, Texas) – the first involving patients who have had a recent (<10 days) traumatic brain injury. They will image using both C1 and C2 pyruvate. So it will be super exciting to see these data! A second trial they're carrying out at UT Southwestern is a healthy brain study, again using C1 and C2 pyruvate. The healthy brain data is obviously vital, as I mentioned at the beginning, and there is also a healthy brain study occurring here at UCSF.
- To support the clinical studies, descriptions of hardware, potential protocols, proof of concept studies, and data analysis methods have begun to be published with respect to human brain data. Work in this area previously applicable to imaging brain cancer patients can obviously be translated to some extent to these neurological diseases, such as work which showed feasibility of metabolic measurements in paediatric brain tumor patients, presented at ISMRM last year. A small published study from the UK in 4 healthy human volunteers showed clearly visible pyruvate, lactate and bicarbonate signal, as shown in this image. They noted more signal overall (of all metabolites) seen in the gray matter than the white matter of the brain. This really highlights again the need to understand the differences between preclinical models and humans given their previous work showing a lack of lactate signal in the pig brain without opening the blood brain barrier, whereas here lactate signal is seen with no intervention.
- One important question that needs answering is when imaging the brain as a whole, do we really understand where the signal is coming from? And, because the answer is not yet, can we work to distinguish which brain cells types are majoritively contributing to the signal of, for example, lactate? But, as with other fields such as cancer, there is great potential for HP MRS to contribute to treatment monitoring in several neurological disorders. If we can overcome the challenges presented, and provide valuable monitoring data, we may discover hints for understanding the underlying mechanistic changes, which may lead to new treatments.