Hyperpolarized 13C MR metabolic imaging can detect neuroinflammation in vivo in a multiple sclerosis murine model

Caroline Guglielmetti; Chloé Najac; Alessandro Didonna; Annemie Van der Linden; Sabrina M Ronen; Myriam M Chaumeil

doi:10.1073/pnas.1613345114

. 2017 Jul 31;114(33):E6982–E6991. doi: 10.1073/pnas.1613345114

Hyperpolarized ¹³C MR metabolic imaging can detect neuroinflammation in vivo in a multiple sclerosis murine model

Caroline Guglielmetti ^a,^b, Chloé Najac ^c, Alessandro Didonna ^d, Annemie Van der Linden ^b, Sabrina M Ronen ^c,^e, Myriam M Chaumeil ^a,^c,¹

PMCID: PMC5565402 PMID: 28760957

Significance

Cells from the innate immune system, namely microglia and macrophages (mononuclear phagocytes, MPs), play a central role in the progression of neurological disorders such as multiple sclerosis. Such cells can contribute to lesion formations (proinflammatory) or participate in remyelinating processes (neuroprotective). When differentiated to a proinflammatory phenotype, MPs experience metabolic reprogramming leading to increased glycolysis and production of lactate. In this study we showed that a new metabolic imaging method, namely ¹³C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized pyruvate, can detect increased lactate production from proinflammatory MPs, a mechanism mediated by pyruvate dehydrogenase kinase 1 upregulation, in a preclinical model of multiple sclerosis. These findings validate the potential of ¹³C MRSI of hyperpolarized pyruvate for in vivo detection of neuroinflammation.

Keywords: hyperpolarized ¹³C MR spectroscopy, multiple sclerosis, neuroinflammation, metabolism, macrophages

Abstract

Proinflammatory mononuclear phagocytes (MPs) play a crucial role in the progression of multiple sclerosis (MS) and other neurodegenerative diseases. Despite advances in neuroimaging, there are currently limited available methods enabling noninvasive detection of MPs in vivo. Interestingly, upon activation and subsequent differentiation toward a proinflammatory phenotype MPs undergo metabolic reprogramming that results in increased glycolysis and production of lactate. Hyperpolarized (HP) ¹³C magnetic resonance spectroscopic imaging (MRSI) is a clinically translatable imaging method that allows noninvasive monitoring of metabolic pathways in real time. This method has proven highly useful to monitor the Warburg effect in cancer, through MR detection of increased HP [1-¹³C]pyruvate-to-lactate conversion. However, to date, this method has never been applied to the study of neuroinflammation. Here, we questioned the potential of ¹³C MRSI of HP [1-¹³C]pyruvate to monitor the presence of neuroinflammatory lesions in vivo in the cuprizone mouse model of MS. First, we demonstrated that ¹³C MRSI could detect a significant increase in HP [1-¹³C]pyruvate-to-lactate conversion, which was associated with a high density of proinflammatory MPs. We further demonstrated that the increase in HP [1-¹³C]lactate was likely mediated by pyruvate dehydrogenase kinase 1 up-regulation in activated MPs, resulting in regional pyruvate dehydrogenase inhibition. Altogether, our results demonstrate a potential for ¹³C MRSI of HP [1-¹³C]pyruvate as a neuroimaging method for assessment of inflammatory lesions. This approach could prove useful not only in MS but also in other neurological diseases presenting inflammatory components.

Multiple sclerosis (MS) is a multifaceted disorder of the CNS and is one of the most common causes of neurological disability in young adults (1, 2). Cortical and white-matter demyelination occurs early in MS pathogenesis and has been associated with disease progression and subsequent cognitive impairment (3). In the vast majority of cases, demyelinating lesions present a high inflammatory component, with elevated density of activated microglia/macrophages (mononuclear phagocytes, MPs) (4–6). Importantly, evidence suggests that proinflammatory MPs are one of the most abundant sources of reactive oxygen species (7), which mediate demyelination and axonal injury (8). Due to their central role in MS pathogenesis, noninvasive imaging of proinflammatory MPs would be of high importance for monitoring progression and response to antiinflammatory therapeutic approaches.

Several recent studies have demonstrated that, upon activation and differentiation toward a proinflammatory phenotype, MPs undergo metabolic reprogramming toward enhanced glucose uptake and increased glycolysis (9–14). As for the Warburg effect observed in cancer cells, this augmented glycolysis takes place under aerobic conditions and results in the majority of pyruvate being converted to lactate (15). However, the molecular mechanisms by which aerobic glycolysis is increased in MPs are still not fully understood. Novel studies have uncovered a potential underlying mechanism, showing pyruvate dehydrogenase kinase 1 (PDK1) up-regulation in proinflammatory MPs (13, 16). Because PDK1 inhibits pyruvate dehydrogenase (PDH), the enzyme that controls pyruvate entrance into the Krebs cycle, PDK1 up-regulation in activated MPs results in increased pyruvate-to-lactate conversion (12, 17, 18). In line with these observations, lactate has long been suggested as an imaging marker of neuroinflammation in the clinic. A few studies on MS patients have used ¹H magnetic resonance spectroscopy (MRS)/MRS imaging (MRSI) approaches and detected increased levels of ¹H lactate in gadolinium-enhancing lesions (19, 20) as well as in the cerebrospinal fluid of patients with such active lesions (21–24).

Over the last decade, dissolution dynamic nuclear polarization coupled with ¹³C MRSI has developed as a metabolic imaging approach of high potential (25, 26). Through increase in the MR-detectable signal of ¹³C compounds by up to 50,000 [so-called hyperpolarized (HP) probes] this methodology has enabled the noninvasive monitoring of several metabolic reactions in vivo (27–32). In particular, in the context of cancer, HP ¹³C MRSI has been shown to improve the noninvasive detection of cancerous lesions through the monitoring of the increased HP [1-¹³C]pyruvate-to-lactate conversion, not only in preclinical cancer models but also in the clinical setting (33).

To date, ¹³C MRSI of HP [1-¹³C]pyruvate has only been applied to investigate inflammation in three studies: a rat model of radiation-induced lung injury (34), a mouse model of inflammatory arthritis (35), and a mouse model of acute liver injury (36). In all three cases, ¹³C MRSI of HP [1-¹³C]pyruvate was capable of detecting an increase in the HP [1-¹³C]lactate-to-pyruvate ratio in the inflammatory region, which was associated with an increased number of macrophages as detected by H&E staining. These three studies are in line with the literature of increased glycolysis in activated MPs. However, to date, no studies have reported the use of ¹³C MRSI of HP [1-¹³C]pyruvate to study neuroinflammation, and underlying biochemical mechanisms linked to increased HP [1-¹³C]lactate production in inflammatory models are still lacking.

In this context, we questioned whether ¹³C MRSI of HP [1-¹³C]pyruvate could monitor the presence of activated MPs in neuroinflammatory lesions in the mouse brain. We conducted a longitudinal study of the toxin-induced cuprizone (CPZ) mouse model, a well-established MS model characterized by high spatial and temporal reproducibility of neuroinflammatory lesions (37, 38). Our results demonstrated a significant increase in HP [1-¹³C]pyruvate-to-lactate conversion in the corpus callosum of CPZ mice as detected by ¹³C MRSI at 14.1 T. Using immunofluorescence, we then showed that increased HP [1-¹³C]lactate production was associated with a high density of activated proinflammatory MPs that overexpressed PDK1. We further showed that the enzymatic activity of PDH was significantly down-regulated in neuroinflammatory lesions, likely due to PDK1 up-regulation, thus providing a putative mechanism for the increased HP [1-¹³C]lactate production observed by ¹³C MRSI. Finally, using transgenic mice exhibiting an impaired microglial function following CPZ diet, we further confirmed that the increased HP [1-¹³C]lactate conversion was primarily driven by the presence of activated MPs in cerebral lesions.

Results

Histological and MRI Characterization of the CPZ Mouse Model.

In the initial part of this study our goal was twofold. First, we wanted to confirm that, in our hands, the CPZ mouse model was behaving as previously described in terms of spatial and temporal distribution of lesions (37, 39–42). Next, we evaluated the potential of T₂-weighted MRI at high field strength (14.1 T) to detect cerebral lesions in CPZ mice.

As shown in Fig. 1 A–C, histological analyses confirmed our CPZ model reproduced findings reported in the literature, presenting demyelination (myelin basic protein, MBP), recruitment of MPs (ionized calcium-binding adapter molecule 1, Iba-1) into the corpus callosum, accumulation of oligodendrocytes progenitor cells (OPCs) (PDGF receptor alpha chain, PDGFR-α), and astrogliosis (glial fibrillary acid protein, GFAP). After 4 wk (W4) of CPZ diet, myelin content was significantly decreased by 86 ± 9% of control (CTRL) level (P < 0.0001) and MP number significantly increased by a dramatic 5,230 ± 762% (P < 0.0001). This massive infiltration of MPs after W4 of CPZ was mainly restricted to the corpus callosum and surrounded the dorsal hippocampal formation (Fig. 1B). In contrast, at 6 wk (W6) of CPZ diet, remyelination reached 51 ± 23% of the CTRL level and MPs were strongly diminished to 30 ± 6% of W4 (P = 0.0285 and P < 0.0001, respectively). At the end of the recovery period both myelin content and MP numbers reached CTRL levels. In addition, significant accumulation of OPCs (PDGFR-α) into the corpus callosum was observed at W4 of CPZ (668 ± 500%, P = 0.0299) and was later followed by a decrease to CTRL levels once remyelination had occurred at W6 + W6 recovery (P = 0.0479). Astrogliosis, as demonstrated by an increase of GFAP, was present after W4 of CPZ (362 ± 200%, P = 0.0004), peaked after W6 of CPZ (645 ± 200%, P < 0.0001), and remained elevated after the recovery period, eventually forming a glial scar (410 ± 225%, P < 0.0001). When looking at all cell types it is important to note that the maximum level of MP staining reached 83 ± 2% area at W4 (Iba-1+), whereas the maximum level of astrocytes reached 54 ± 3% area at W6 (GFAP+), and OPC staining was maximum at W4 with 2.2 ± 1% area (PDGFR-α+).

On T₂-weighted MR images, hyperintensity could be observed in the corpus callosum of CPZ mice following W4 and W6 of CPZ administration (Fig. 1D, arrows). Upon quantification, our results showed that normalized T₂-weighted (nT₂w) signals from the corpus callosum were significantly different between CPZ and CTRL groups (P < 0.0001 for group and time effects and for group and time interaction). The nT₂w signals from the corpus callosum of CPZ-treated mice were significantly higher than those from the CTRL mice at any time point following CPZ diet (P < 0.0001 for W4, W6, and W6 + W6 recovery). Furthermore, nT²w signals from CPZ mice were increased by 42 ± 13% after W4, 33 ± 16% after W6 of CPZ diet, and 12 ± 9% after recovery compared with W0 (P < 0.0001 for W4 and W6 and P = 0.0174 for W6 + W6 recovery). However, no significant difference in nT₂w signal values could be observed between W4 and W6 CPZ (P = 0.0562), despite the differences in underlying pathophysiological events observed at these two time points. In the CTRL group, nT₂w signals significantly decreased over time (P = 0.0269), likely reflecting myelination of the corpus callosum in young adult mice (40, 43).

Increased Production of HP [1-¹³C]Lactate Following MP Activation in CPZ-Induced Lesions.

Next, we evaluated the potential of ¹³C MRSI of HP [1-¹³C]pyruvate to monitor the presence of proinflammatory MPs. To do so, as shown in Fig. S1, MR experiments were performed at W0, W4, and W6 of CPZ diet and W6 of CPZ diet followed by W6 of standard rodent chow.

As shown in Fig. 2 A and B, the level of HP [1-¹³C]pyruvate detected in the corpus callosum voxel remained stable over time in CTRL and CPZ mice. In contrast, a significant increase of HP [1-¹³C]lactate was observed in the corpus callosum region following W4 of CPZ administration (Fig. 2A). Upon full quantification, our results showed that the ratio of HP [1-¹³C]lactate to pyruvate was significantly different between CPZ and CTRL groups over time (P = 0.0014 for group effect, P = 0.045 for time effect, and P = 0.0004 for group and time interaction). Specifically, when comparing CTRL and CPZ groups, the HP [1-¹³C]lactate-to-pyruvate ratio was more than threefold higher in the corpus callosum of CPZ mice after W4 of CPZ diet compared with age-matched CTRL (P = 0.0006), a time point that corresponds to the highest level of activated proinflammatory MPs (Fig. 2C). At that time point, T₁-weighted MRI after injection of a gadolinium-based contrast agent showed similar signal enhancement in the corpus callosum of CTRL and W4 CPZ mice (Fig. S2). Next, longitudinal evaluation revealed a significant increase of the HP [1-¹³C]lactate-to-pyruvate ratio after W4 of CPZ compared with W0 (96 ± 74% of W0, P = 0.0002), followed by a significant decrease after W6 of CPZ (42 ± 59% of W4, P = 0.0194), when MPs numbers are strongly decreased and remyelination is occurring. By the end of the recovery period, the HP [1-¹³C]lactate-to-pyruvate ratio remained significantly decreased compared with W4 CPZ (131 ± 24% of W4, P = 0.0346) and was no longer significantly different from W0 values (P = 0.2005) (Fig. 2D). The two animals that were not imaged at W6 + W6 were excluded from the statistical analysis between W4 and W6 + W6.

Fig. S2. — Contrast-enhanced MRI following CPZ diet. (A) Representative T₁-weighted MR images of a CTRL mouse and a mouse at W4 CPZ, prior to (pre-contrast) and after (post-contrast) injection of a gadolinium-based contrast agent (arrow indicates the time of contrast agent injection), showing characteristic enhancement of surrounding tissues and blood vessels on postcontrast images. Time courses of signal intensity (B) for the corpus callosum and the thalamus, showing an increase in both regions following contrast agent injection in CTRL and W4 CPZ mice. Quantitative analyses of the area under the curve (C), slope (D), and relative signal enhancement (E) showed no significant differences between CTRL and W4 CPZ mice (n = 3 mice per group). All values are reported as mean ± SD, except D, which displays mean ± SEM. AU, arbitrary unit.

As shown on the heat maps of HP [1-¹³C]lactate-to-pyruvate ratio, the increase in HP [1-¹³C]lactate production was localized to the corpus callosum voxel only, in line with the high level of activated MPs in that cerebral region (Fig. 3A). To further assess the specificity of ¹³C MRSI of HP [1-¹³C]pyruvate to detect proinflammatory MPs, we examined the levels of HP [1-¹³C]lactate and HP [1-¹³C]pyruvate in a thalamic voxel (dark blue) as well as in an heterogeneous neck voxel outside the brain (light blue), regions that do not display a large recruitment, proliferation, and activation of MPs (Fig. 3B). In line with the HP [1-¹³C]lactate maps, the grid of the HP ¹³C spectra showed an increased HP [1-¹³C]lactate in the region that contains the demyelinated and inflamed corpus callosum (Fig. 3B, red) but not in the adjacent brain regions (Fig. 3B, dark and light blue). In both the thalamic and the neck region the level of HP [1-¹³C]pyruvate remained stable over time for both CPZ and CTRL groups (Fig. 3 C and D) and the HP [1-¹³C]lactate-to-pyruvate ratio did not significantly differ between CPZ and age-matched CTRL (Fig. 3 E and F). T₁-weighted postcontrast also showed no differences in thalamus kinetic parameters between CTRL and W4 CPZ mice (Fig. S2).

Fig. 3. — Increased HP [1-¹³C]lactate-to-pyruvate ratio is restricted to the inflamed and demyelinated corpus callosum. (A) HP [1-¹³C]lactate-to-pyruvate ratio heat maps display an increased HP [1-¹³C]lactate production after W4 of CPZ diet in the corpus callosum only. (B) T₂-weighted image overlaid with the grid used for HP ¹³C MR acquisition is shown for a W4 CPZ mouse. The corresponding sum of dynamic spectra is presented. Increased HP [1-¹³C]lactate can be observed in the voxel containing the corpus callosum (red) but not in a voxel containing the thalamic region (dark blue) or in a nonbrain voxel located beneath the brain (neck voxel, light blue). (C and D) HP [1-¹³C]pyruvate delivery as well as (E and F) HP [1-¹³C]lactate-to-pyruvate ratios were not significantly different between CTRL and CPZ groups over time in the thalamic and neck region (n = 5–7 mice per group; C and E thalamus and D and F neck). All values are reported as mean ± SD. A.U., arbitrary unit.

The Increase in HP [1-¹³C]Lactate Signal Is Associated with Up-Regulation of PDK1 in Activated MPs and Decreased PDH Activity in the Corpus Callosum.

We performed immunofluorescence for PDK1, a protein that inhibits PDH and thus controls pyruvate entrance into the Krebs cycle. Interestingly, we demonstrated a significant up-regulation of PDK1 (PDK1+) in activated MPs (Iba-1+) following W4 of CPZ administration in the corpus callosum, but not in other brain regions (cortex and thalamus), as shown in Fig. 4A and Fig. S3. Whereas only 12 ± 7% of Iba-1+ were also PDK1+ at W0, this number significantly increased at W4 (87 ± 7% of MPs are PDK1+/Iba-1+, P < 0.0001) and W6 after CPZ diet (82 ± 8% of MPs are PDK1+/Iba-1+, P = 0.001), in line with a higher density of activated MPs at these time points. Cellular colocalization of PDK1 and Iba-1 was further confirmed by confocal microscopy (Fig. 4B). Importantly, GFAP/PDK1 and PDGFR-α/PDK1 costaining revealed that neither astrocytes nor OPCs overexpressed PDK1 at any time point (Fig. 4B and Fig. S3), providing additional evidence that these other cell types do not play a major role in the HP [1-¹³C]lactate signal increase at W4 of CPZ.

Fig. 4. — Increased HP [1-¹³C]lactate production is associated with up-regulation of PDK1 in activated MPs and decreased PDH activity in the corpus callosum. (A) Immunofluorescence staining for PDK1 (green) and MPs (Iba-1, red) reveal overexpression of PDK1 in MPs after W4 of CPZ diet (yellow). Quantitative analyses confirmed the significant up-regulation of PDK1 after W4 of CPZ (P < 0.0001), (n = 3 mice per time point). (Scale bar: 100 µm.) (B) Confocal microscopy confirmed cellular colocalization (yellow, arrows) of PDK1 (green) in MPs (Iba-1, red) at W4 of CPZ diet. In contrast, astrocytes (GFAP, red, arrow) and OPCs (PDGFR-α, red, arrow) staining did not colocalize with PDK1 staining (green) at W4 of CPZ diet, as shown by confocal microscopy. (Scale bar: 10 µm.) (C) Quantitative analyses revealed a significant decrease of PDH enzyme activity at W4 of CPZ diet (P = 0.0026) and (D) no significant change in LDH-A enzyme activity (reported as V_max). (E) Serum lactate levels were significantly increased at W4 and W6 of CPZ diet (161 ± 62% W4 P = 0.0014; 166 ± 48% W6 P = 0.0007) and returned to CTRL levels by the end of the recovery period (P = 0.0024), (n = 4–6 mice per time point). All values are reported as mean ± SD (one-way ANOVA, Tukey HSD post hoc test **P < 0.01, ***P < 0.001).

Fig. S3. — Immunofluorescence staining of the corpus callosum, thalamus, and cortex following CPZ diet and evaluation of PDH activity in the thalamus. (A) Representative immunofluorescence images of MPs (Iba-1, red) and PDK1 (green) in the thalamus and cortex showing no expression of PDK1, and hence no colocalization with Iba-1 at any time point of interest (n = 3 mice per group). (Scale bar: 100 µm.) (B) Representative immunofluorescence staining of astrocytes (GFAP, red) and OPCs (PDGFR-α, red) showing no colocalization with PDK1 staining (green) in the corpus callosum at any time points of interest (n = 3 mice per group). (Scale bar: 100 µm.) (C) Quantitative analyses of PDH enzyme activity in the thalamus revealed no significant changes following CPZ diet (n = 4–7 mice per group). (D) Immunofluorescence staining for LDH-A and nuclear staining (Hoechst) show that LDH-A is not detected in the corpus callosum following W4 of CPZ diet. LDH-A expression was found in the CTRL area CA-1 of the hippocampus (n = 3 mice per group). (Magnification: 20×.)

Next, we wanted to investigate whether PDK1 up-regulation by Iba-1 MPs was associated with a modulation in the activity of its downstream target PDH. As shown in Fig. 4C, PDH activity was significantly decreased in the corpus callosum of W4 mice compared with W0 (57 ± 15% decrease, P = 0.0026), but not in the thalamus (Fig. S3), suggesting a regional effect of Iba-1+ MPs PDK1 up-regulation on PDH activity. Further in line with this putative link, after W6 of CPZ and after the recovery period the number of PDK1+/Iba-1+ MPs present in the corpus callosum significantly decreased and PDH activity no longer differed from CTRL levels. We also investigated lactate dehydrogenase A (LDH-A), the isoenzyme that converts pyruvate to lactate and is overexpressed in most cancer types. Unlike in cancer, we did not observe an up-regulation of LDH-A staining in the corpus callosum (Fig. S3) or a change in LDH-A activity at any time point (Fig. 4D), thus ruling out overexpression of this enzyme as a main player in the observed increased HP [1-¹³C]lactate production at W4 CPZ.

Finally, we measured serum lactate levels in CPZ and CTRL mice at all time points. As shown in Fig. 4E, serum lactate levels were significantly increased at W4 and W6 of CPZ (161 ± 62% W4 P = 0.0014; 166 ± 48% W6 P = 0.0007) and returned to CTRL levels by the end of the recovery period (P = 0.0024), confirming the systemic effect of CPZ diet (38, 44, 45).

HP [1-¹³C]Lactate Levels Are Not Increased in the Corpus Callosum of CX₃CR1^GFP/GFP Transgenic Mice Harboring Microglial Activation Deficiency, Suggesting a Central Role of MPs to the Detected HP [1-¹³C]Lactate Signal.

To confirm that the production of HP [1-¹³C]lactate was driven by the presence of MPs into the corpus callosum following CPZ diet, we studied CX₃CR1^GFP/GFP mice, which exhibit highly reduced or absent microgliosis, astrogliosis, and demyelination at W4 of CPZ diet, as previously described (39). In these mice, the CX₃CR1 receptor, which is highly expressed in microglia, is replaced by GFP, allowing one to easily monitor microglia levels by immunofluorescence (46).

As shown in Fig. 5A, CPZ diet did not induce hyperintensities characteristic of demyelination and inflammation on T₂-weighted MR images in the corpus callosum of CX₃CR1^GFP/GFP mice. Quantitative analyses of the nT₂w values from the corpus callosum showed that nT₂w were significantly decreased at W4 in CPZ and CTRL CX₃CR1^GFP/GFP, as well as in CTRL nontransgenic mice, reflecting normal myelination throughout development (40, 43). In contrast to nontransgenic CPZ mice, however, no differences in T₂w MR contrast could be detected between CTRL and CPZ groups at W0 or W4. Immunofluorescence analyses further confirmed the absence of microgliosis (CX₃CR1^GFP/GFP labeled microglia), cell infiltration, demyelination, and astrogliosis (Fig. S4) in the corpus callosum of CX₃CR1^GFP/GFP mice, despite W4 of CPZ diet, and in line with previous reports (37, 39).

Fig. S4. — Immunofluorescence staining and HP [1-¹³C]pyruvate delivery in CX₃CR1^GFP/GFP mice following W4 of CPZ diet. (A) Representative immunofluorescence staining for cell infiltration (Hoechst, blue), myelin (MBP, red), astrocytes (GFAP, red), and MPs (Iba-1, red) shows no evident changes between CX₃CR1^GFP/GFP mice that received a CTRL chow or W4 of CPZ diet. (Scale bar: 100 µm.) HP [1-¹³C]pyruvate levels in CX₃CR1^GFP/GFP were not significantly different between CTRL and CPZ groups over time in (B) the corpus callosum, (C) the thalamic area, and (D) the neck region (n = 6 mice per group). All values are reported as mean ± SD. A.U., arbitrary unit.

Importantly, PDH (Fig. 5B) and LDH-A (Fig. 5C) activities measured from the corpus callosum of CX₃CR1^GFP/GFP mice were not significantly different between CTRL and CPZ animals at W4. As expected however, and similar to the nontransgenic model, the serum lactate levels (Fig. 5D) were increased by 173 ± 79% (P = 0.0079) in CX₃CR1^GFP/GFP mice after W4 of CPZ diet compared with CTRL mice, confirming the systemic effect of the CPZ diet.

We then performed HP ¹³C MRSI to further confirm that increased HP [1-¹³C]lactate was associated with the presence of proinflammatory MPs. HP ¹³C spectra acquired in the corpus callosum of CX₃CR1^GFP/GFP mice show no evident differences between CTRL and CPZ groups (Fig. 5E). Spectra quantification showed that, in contrast to the nontransgenic mice, HP [1-¹³C]lactate-to-pyruvate ratios for the corpus callosum were similar between CTRL and CPZ groups at W4 of CPZ diet, in line with the absence of MP activation and infiltration in this transgenic model (Fig. 5F). Similarly, HP [1-¹³C]lactate-to-pyruvate ratios from the thalamic area (Fig. 5G) and the neck (Fig. 5H) were not significantly different between CTRL and CPZ groups at W4. HP [1-¹³C]pyruvate delivery was also unchanged in all regions (Fig. S4).

PDK1 Is Up-Regulated in Activated MPs in Experimental Autoimmune Encephalomyelitis Lesions.

Finally, to further assess the validity of our findings, we investigated whether the up-regulation of PDK1 by MPs was also present in demyelinated lesions in another animal model for MS. For this, we performed immunofluorescence analyses on tissues from experimental autoimmune encephalomyelitis (EAE) mice. Most neuroinflammatory lesions were found in the spinal cord and brainstem in close vicinity to blood vessels. We found that 45 ± 8% of MPs present in the EAE group expressed PDK1 in the spinal cord (Fig. 6 A–C), 28 ± 8% in the brainstem, and 25 ± 7% in the cerebellar white matter (Fig. 6 B and C), whereas the level of detection for PDK1 was below detection for age-matched CTRL (Fig. 6A).

Fig. 6. — Detection of PDK1-expressing MPs after EAE induction. (A) Immunofluorescence staining for MPs (Iba-1, red) shows increased number of MPs and expression of PDK1 (green) in the spinal cord 30 d after EAE induction compared with age- and sex-matched CTRL. (B and C) Quantitative analyses reveal that 45 ± 8% of MPs present in the lesions coexpress Iba-1 and PDK1 (yellow) in the spinal cord, 28 ± 8% in the brainstem, and 26 ± 7% in the cerebellum, whereas the level of PDK1 in the CTRL group was below detection. All values are reported as mean ± SD (n = 3 mice).

Discussion

In this study we applied a metabolic imaging method, ¹³C MRSI of HP [1-¹³C]pyruvate, to the longitudinal study of the CPZ model of MS. HP [1-¹³C]pyruvate-to-lactate conversion increased in the corpus callosum of CPZ mice at W4 of CPZ diet, a time point presenting the highest density of proinflammatory MPs. In contrast, in knockout CX₃CR1^GFP/GFP animals with low microglial activation HP [1-¹³C]lactate levels were not increased, suggesting a central role for MPs in the detected HP signal. We further showed that, at W4 in the nontransgenic model, the majority of activated MPs present in the corpus callosum overexpressed PDK1 and that PDH activity was significantly decreased in this region, providing a plausible mechanism for the observed increased HP [1-¹³C]lactate levels.

We opted to investigate the well-established CPZ mouse model for MS, whose strengths lie in the localization of the lesions, their well-known pathogenesis, and their reproducibility in time and space, allowing for longitudinal validation of imaging methods (37, 47, 48). In this model, the vast majority of activated MPs are endogenous microglia, not peripheral macrophages (49–52). At W4 of CPZ, proinflammatory MPs in the corpus callosum express the phagocytic marker CD11b and are responsible for the phagocytosis of myelin debris (53, 54). This supportive action of MPs is an essential step for subsequent remyelination at W6 and W6 + W6 recovery (49, 55). Indeed, directing MPs toward an antiinflammatory phenotype before and during the first weeks of CPZ diet prevents in part oligodendrocyte cell death, demyelination, and astrogliosis (52, 56). We chose to monitor CPZ mice at W4 and W6 of CPZ diet and after a subsequent period of 6 wk without CPZ because these three time points are characterized by different cellular events. At W4 of CPZ diet, cerebral lesions show a large recruitment and proliferation of MPs, which induce demyelination and can reach up to 50 times the density of microglia present under normal conditions (40). By W6 of CPZ diet, most of the MPs have disappeared and remyelination is ongoing despite continuous administration of the toxin (57, 58). After the 6-wk recovery period, remyelination has occurred, MP number has returned to baseline, and only astrogliosis remains. These marked differences between the cellular events happening at W4, W6, and W6 + W6 recovery justifies the longitudinal follow-up of CPZ mice at these three time points and enabled us to evaluate the potential of ¹³C MRSI of HP [1-¹³C]pyruvate to detect the presence of proinflammatory MPs.

Current clinical evaluation of MS brain lesions essentially includes conventional T₂-weighted and gadolinium-enhanced T₁-weighted MRI (59, 60). Although these methods provide high spatial sensitivity, prediction of outcome and response to treatment remains challenging. Gadolinium-enhanced T₁ lesions are indicative of altered blood–brain barrier (BBB) and are considered the main radiologic marker of active MS lesions (59, 61). However, whereas density of active lesions are linked to treatment efficacy in some cases, formation of new T₁ lesions may occur subclinically and thus more frequently than clinical relapses, limiting treatment monitoring (62). Furthermore, noninvasive methods for assessment of neuroinflammatory status in chronic, nonenhancing lesions in gray and white matter are still lacking. Such limitations represent a major drawback for evaluation of lesion formation and monitoring of regenerative and antiinflammatory therapies. In this study we showed that, whereas T₁-weighted postcontrast MRI confirmed the lack of BBB breakdown in the CPZ model, nT₂w values enabled the detection of CPZ-induced lesions and long-lasting changes associated with a demyelinating event. However, this measure could not readily distinguish between lesion stages linked to different underlying cellular events, notably acute inflammation at W4 CPZ. In contrast, using ¹³C MRSI of HP [1-¹³C]pyruvate, we were able to detect an increase of HP [1-¹³C]lactate production specifically at W4 CPZ in lesions presenting a high level of proinflammatory MPs, thus highlighting the potential added value of this metabolic imaging strategy compared with conventional MR methods.

Following i.v. injection, we detected delivery of HP [1-¹³C]pyruvate in the corpus callosum of CTRL and CPZ mice. Interestingly, levels of HP [1-¹³C]pyruvate were comparable between these two groups for all time points. This result is in line with the fact that, unlike in the EAE model, the induction of lesions following CPZ diet occurs without BBB disruption (49–51, 58, 63). Furthermore, and importantly, we were able to detect production of HP [1-¹³C]lactate from HP [1-¹³C]pyruvate in the corpus callosum of both CTRL and CPZ animals and a significant increase in HP [1-¹³C]lactate production at W4 in the CPZ group only. In agreement with other HP ¹³C MRSI studies (32), this result suggests that, even in the presence of an intact BBB, enough HP [1-¹³C]pyruvate enters the brain for the conversion of HP [1-¹³C]pyruvate to lactate and its variations with disease progression to be detected during the time frame of an experiment.

To assess whether changes in endogenous lactate levels and/or vascular changes contributed to the increased HP [1-¹³C]lactate signal observed, through label exchange into the lactate pool (32, 64), we performed T₁-weighted MRI and serum lactate level analysis. T₁-weighted MRI postcontrast showed comparable kinetics of signal enhancement in the corpus callosum of CTRL and CPZ animals at W4, suggesting that global vascular changes (blood flow, permeability, and perfusion) are unlikely to be the driving mechanisms behind the detected increase in HP [1-¹³C]lactate production observed at that time point. Regarding lactate levels, our results show that serum lactate levels are significantly increased following W4 of CPZ diet, in both nontransgenic and CX₃CR1^GFP/GFP animals. Importantly, however, only in the nontransgenic animals presenting a high density of MPs in the corpus callosum did the HP [1-¹³C]lactate significantly increase at W4. Furthermore, this increase was specific to the corpus callosum of the nontransgenic mice only and was not observed in thalamus or in the neck voxel containing blood vessels in any of the two models. Finally, albeit increased compared with CTRL, serum lactate levels in nontransgenic CPZ animals were highly comparable between W4 and W6 of CPZ diet (P = 0.986), whereas HP [1-¹³C]lactate signal in the corpus callosum was only increased at W4 and not at W6. Altogether, these data strongly support the fact that MP proliferation in the corpus callosum at W4 is likely responsible for the increased HP [1-¹³C]lactate signal seen in the DNP experiments. However, one cannot rule out the fact that inflammation-related increases in the flow of blood containing ¹³C-labeled lactate rather than metabolic processes within MPs can contribute to the detected changes in HP [1-¹³C]lactate, because post-Gd T₁-weighted MR data cannot rule out this possibility. Additionally, some contribution of endogenous brain lactate (e.g., from necrotic regions), albeit unlikely, cannot be ruled out.

We showed that MPs, astrocytes, and OPCs were modulated by CPZ diet in the corpus callosum of nontransgenic mice, with a dramatic 50-fold increase in MP density in this region at W4. We also demonstrated that only proinflammatory MPs up-regulate the enzyme PDK1, whereas astrocytes and OPCs did not. This result, combined with the lower levels of OPCs and the different temporal evolution of astrogliosis (maximum at W6), highly suggest that PDK1+ MPs are the major cell type contributing to the increased HP [1-¹³C]lactate at W4 (40). Interestingly, we found a global decrease of PDH activity in the corpus callosum of nontransgenic CPZ mice at W4, but not in the thalamus, whereas LDH-A activity in both regions was unchanged by CPZ diet. In transgenic CX₃CR1^GFP/GFP animals with low microglial activation both PDH and LDH-A were unchanged in the corpus callosum. These findings provide a plausible explanation for the increased HP [1-¹³C]lactate observed at W4 of CPZ diet in nontransgenic mice only, with low PDH activity preventing the entrance of pyruvate into the Krebs cycle and redirecting HP [1-¹³C]pyruvate toward HP [1-¹³C]lactate production. These results also suggest that the underlying mechanism of increased HP [1-¹³C]lactate production linked to inflammation is different from the one observed in cancer, in which LDH-A overexpression is the most common player (32, 65). Future studies performed in in vitro controlled systems (e.g., MP cell cultures) are now needed to fully confirm the link between PDK1 up-regulation, PDH activity, and increased lactate production. Nonetheless, our results are in line with recent studies (13, 16) and provide a strong rationale for the observed increase in HP [1-¹³C]lactate. We additionally showed the expression of PDK1 in MPs in EAE-induced inflammatory and demyelinated spinal cord lesions (Fig. 6), suggesting that PDK1 up-regulation is a commonly shared feature of proinflammatory MPs that is conserved among different neuroinflammation models.

From a technical perspective, we evaluated the dynamic conversion of HP [1-¹³C]pyruvate into lactate by acquiring spectra every 3 s for a period of a minute. This achievement comes with the trade-off of a lower spatial resolution, which may result in lower sensitivity to subtle lactate changes. For instance, after W6 of CPZ administration we observed a strong reduction of MPs in the corpus callosum. Although most of the remaining MPs still express PDK1 and there is a trend toward decreased PDH activity compared with CTRL, we did not detect a significantly increased production of HP [1-¹³C]lactate at that time point, which may be explained by the limited sensitivity of HP ¹³C MRSI to lower MP number. Furthermore, the lack of increase in the HP [1-¹³C]lactate-to-pyruvate ratios in two of seven mice at W4 (Fig. 2D), despite the likely infiltration of MPs in these animals, should be acknowledged as a probable technical failure of the DNP methodology. Future improvements in hardware and MR acquisition schemes will likely help in reaching a higher sensitivity/specificity and improve the detection of smaller lesions.

In conclusion, this paper shows that metabolic imaging using HP ¹³C MRSI can be used to monitor neuroinflammatory lesions noninvasively in a preclinical model for MS. Increase in HP [1-¹³C]lactate production can be detected in cerebral lesions and is associated with high levels of PDK1+-activated MPs and subsequent regional PDH inhibition. Altogether, our results demonstrate a potential for ¹³C MRSI of HP [1-¹³C]pyruvate as a neuroimaging method for assessment of inflammatory lesions. Because HP ¹³C MRSI is clinically translatable (33) and expanding rapidly, this study is of high significance for future clinical trials not only on MS but also all neurological diseases presenting an inflammatory component (66). When used in conjunction with conventional MRI techniques, such a method would enhance diagnosis and help refine therapeutic regimens, which will ultimately improve clinical outcome and patient care.

Materials and Methods

Animals and Experimental Outline.

All animal research was approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco. For CPZ studies, 8-wk-old B6(Cg)-Tyr^c-2J/J (stock no. 000058) albino female mice and 8-wk-old B6.129P-Cx3cr1^tm1Litt/J (stock no. 005582) mice (referred to above as CX₃CR1^GFP/GFP) were purchased from Jackson Laboratories. Mice [n = 43 B6(Cg)-Tyr^c-2J/J; n = 6 Cx3cr1^tm1Litt/J] received a 0.2% wt/wt CPZ (Sigma-Aldrich) diet for up to 6 wk. Age-matched CTRL mice received a standard rodent chow [n = 22 B6(Cg)-Tyr^c-2J/J; n = 6 Cx3cr1^tm1Litt/J]. For B6(Cg)-Tyr^c-2J/J mice, a subset of animals were imaged longitudinally prior (W0), and after 4 and 6 wk of CPZ (W4 and W6) as well as after 6 wk of CPZ followed by 6 wk of standard rodent chow (W6 + W6 recovery) (n = 7 CPZ and 7 CTRL). For Cx3cr1^tm1Litt/J animals, all mice were imaged at W0 and W4 (n = 6 CPZ and 6 CTRL). For immunofluorescence staining, enzyme activity assays, and serum lactate levels, a subset of mice was killed at each time point (n = 3–6 CTRL, 3–6 W4, 3–6 W6, and 3–6 W6 + W6 recovery). For EAE induction, 8-wk-old C57BL/6J female mice were injected s.c. with 100 µg of myelin oligodendrocyte glycoprotein (MOG_35–55), in complete Freund’s adjuvant (Difco Laboratories) (67). After immunization and 2 d later mice received 400 ng of pertussis toxin i.p. At 30 d postinjection mice (n = 3 EAE and 3 age-matched CTRL) were killed and CNS tissue was collected. For all experiments animals were observed daily, and clinical signs were assessed as follows: 0, no signs; 1, decreased tail tone; 2, mild monoparesis or paraparesis; 3, severe paraparesis; 4, paraplegia; 5, quadraparesis; and 6, moribund or death. Mice with an EAE score of 2.5 ± 0.5 at 30 days postinjection were used for the experiments.

In Vivo MR Acquisitions.

All in vivo MR experiments were conducted on a 14.1 T vertical MR system (Agilent Technologies) equipped with 100 G/cm gradients and a dual tune ¹H-¹³C volume coil (Ø_I = 40 mm). For each imaging session, mice were anesthetized using isoflurane (1–3% in O₂) and a 27-gauge catheter was secured in the tail vein to allow for i.v. injection of the HP probe. Animals were placed in a dedicated cylindrical cradle allowing for reproducible positioning of the mouse head; the cradle was then tightly inserted inside the volume coil, which was secured to the MR bore to ensure similar positioning between experiments. Respiration and temperature were continuously monitored to ensure animal well-being and data reproducibility.

T₂-weighted MRI was acquired using the following parameters: TE/TR = 20/1,200 ms, slice thickness = 0.5 mm, number of averages = 2, matrix = 256 × 256, and field of view (FOV) = 30 × 30 mm². For HP ¹³C MRSI acquisitions, 24 μL of [1-¹³C]pyruvate preparation was hyperpolarized using a Hypersense DNP polarizer (Oxford Instruments) for 1 h (29). Note that all pyruvate preparations used in this study were prepared as large batches and underwent quality control to ensure reproducible polarization level between experiments (10–12%). After dissolution, HP [1-¹³C]pyruvate was rapidly dissolved in isotonic buffer (pH ∼7) to a final concentration of 80 mM. A final volume of 300 μL of the solution was then injected i.v. over 12 s through the tail vein catheter. From the beginning of the iv injection, 2D dynamic chemical shift imaging (CSI) ¹³C data were acquired using the following parameters: TE/TR = 1.2/60 ms; spectral width = 2,500 Hz; 128 points; flip angle (FA) = 10°; FOV = 24 × 24 mm²; matrix 8 × 8 × 8; in-plane resolution 3 × 3 mm²; 5-mm slice thickness; scan time 3 s per time point; and 16 time points (68).

T₁-weighted images (TE/TR = 1.14/39 ms, FA = 40°, 10 slices, thickness = 1.5 mm, NA = 1, matrix = 128 × 128, and FOV = 22.4 × 22.4 mm²) were acquired prior (five images) and after (25 images) i.v. injection of Magnevist (gadopentetate dimeglumine, gadolinium-based contrast agent, 1 mmol/kg).

MR Data Analysis.

The corpus callosum was defined as the region of interest and manually delineated on the T₂-weighted images according to the Franklin and Paxinos anatomical mouse brain atlas (69) with AMIRA software (Mercury Computer Systems). The average signals from the corpus callosum were normalized to the mean cerebrospinal fluid signal of the third ventricle as signal value standard. In a similar manner, the corpus callosum and the thalamus were delineated on T₁-weighted images and simple kinetic parameters of contrast enhancement (area under the curve, slope, and relative signal enhancement ([(MR signal postcontrast − MR signal precontrast)/MR signal precontrast]*100) were calculated, as surrogate values of global perfusion, permeability, and blood flow, as previously described (70, 71).

HP ¹³C MRSI datasets were analyzed using the in-house SIVIC software (https://sourceforge.net/apps/trac/sivic/) and custom-built programs written in MATLAB (MATLAB R2011b; The MathWorks Inc.). HP [1-¹³C]lactate and HP [1-¹³C]pyruvate levels were calculated as the sum of integrals over time and normalized to noise level (Fig. S1). Color heat maps of HP [1-¹³C]lactate-to-pyruvate ratio were generated using a sinc-based interpolation of the ¹³C 2D CSI data to the resolution of the anatomical images using custom-built programs written in MATLAB and SIVIC.

Immunofluorescence Acquisition and Analysis.

All analyses were performed according to previously described procedures (72). Mice were first perfused with an ice-cold 0.9% NaCl solution followed by an ice-cold 4% paraformaldehyde (PFA) solution. Next, brains were dissected and further fixed in 4% PFA for 2 h then dehydrated through a sucrose gradient (2 h at 5%, 2 h at 10%, and overnight at 20%). Afterward, brain tissue was snap-frozen in liquid nitrogen and kept at −80 °C until further processing. Ten-micrometer-thick cryosections were collected. Immunofluorescence staining was performed on brain slides using the following antibody combinations: a primary chicken anti-myelin basic protein (MBP) antibody (AB9348; 1:200 dilution; Millipore) with a secondary donkey anti-chicken DyLight 549 antibody (703-506-155, 1:1,000 dilution; Jackson); a primary rabbit anti-Iba-1 antibody (019-19741, 1:500 dilution; Wako) with a secondary donkey anti-rabbit Alexa Fluor 555 (A31572, 1:1,000 dilution; Invitrogen); a primary mouse anti-PDK1 antibody (AB110025, 1:100 dilution; Abcam) with a secondary goat anti-mouse Alexa Fluor 488 (A11017, 1:100 dilution; Invitrogen); a primary rabbit anti-GFAP (AB7779, 1:500 dilution; Abcam) with a secondary goat anti-rabbit Alexa Fluor 488 (A11008, 1:1,000 dilution; Invitrogen) or with a secondary donkey anti-rabbit Alexa Fluor 555 (A31572, 1:1,000 dilution; Invitrogen); and a primary rat anti-mouse CD140a PDGFR-α (clone APA5, 1:500 dilution; BD Pharmigen) with a secondary goat anti-rat 570 Fab (1:50, 112–297-003; Jackson ImmunoResearch). Slides were counterstained using Hoechst 33342 (H3570, 1:2,000 dilution; Invitrogen). Following staining, sections were mounted using Prolong Gold Antifade (P36930; Invitrogen). Fluorescence image acquisition was performed using an inverted microscope (Ti-E; Nikon). The custom-built epi-illumination optics includes excitation lasers at 405 nm (for DAPI), 488 nm (for eGFP), 561 nm (for mCherry), and 642 nm (For Cy5). The images were recorded with an EMCCD camera (iXon+ DU897E-CS0-BV; Andor). The microscope was controlled using custom software written in Python. Quantitative analyses of immunofluorescence images were performed using NIH ImageJ (v1.46r). The levels of microglia/macrophages (Iba-1), astrocytes (GFAP), OPCs (PDGFR-α), PDK1, and myelin (MBP) were determined based on the image-covering staining and expressed as percentage of the total area (39, 40). For confocal microscopy, images were acquired using a Zeiss LSM780 controlled by Zen software (Zeiss 2014). Only linear adjustments were made during image acquisition.

Spectrophotometric Assay.

A subset of mice were killed at each time point of interest [W0 (CTRL), W4, W6, and W6 + W6 recovery] and brains were rapidly dissected. The corpus callosum was then isolated and snap-frozen and samples were stored at −80 °C until further processing. The enzymatic activity of PDH was assessed using spectrophotometric activity assay kit (ab109902; Abcam), according to manufacturer’s guidelines and normalized to the concentration of protein determined by the Bradford method (n = 4–6 mice per group). The enzymatic activity of LDH-A (V_max) was assessed as previously shown (73) (n = 4–6 mice per group).

Serum Lactate Levels.

A subset of mice was anesthetized at each time point [W0 (CTRL), W4, W6, and W6 + W6 recovery] and blood was collected through the tail vein. Serum lactate levels were measured using an assay kit (l-lactate assay kit, colorimetric, ab65331; Abcam) (n = 4–6 mice per group).

Statistical Analysis.

For all experiments sample sizes were determined based on previous experience of tumor-bearing mice imaging using ¹³C MRSI of HP [1-¹³C]pyruvate at 14.1 T (32, 68). Based on this experience, we assumed that seven to eight animals per experimental group studied longitudinally would suffice to get significant HP data using a two-way repeated measure ANOVA test with P = 0.05. For immunohistochemistry and activity assays we determined that three to four animals per group would suffice to get significant data using a one-way ANOVA test with P = 0.05. With such group sizes, we would have 80–90% power to detect an effect size of 1.25.

Results are expressed as mean ± SD. Two-Way ANOVA was used to determine statistical significance of T₂-w MRI and HP ¹³C MRSI between CPZ and CTRL groups and P values were corrected for multiple testing using the Tukey honest significant difference (HSD) post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). To compare nonlongitudinal data between W6 + W6 recovery and all other time points (W0, W4, and W6) unpaired t test was used (^#P < 0.05, ^##P < 0.01, ^###P < 0.001). Additionally, a repeated measures ANOVA, with P values corrected for multiple testing using the Tukey HSD post hoc test, was used to evaluate a statistical significance in the CTRL group (^†P < 0.05, ^††P < 0.01, ^†††P < 0.001). Statistical analyses of immunofluorescence, enzyme activity assays, and serum lactate levels between W0, W4, W6, and W6 + W6 recovery were performed using a one-way ANOVA, and the given P values were corrected for multiple testing using the Tukey HSD post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). To evaluate statistical significance of immunofluorescence, enzyme activity assays and serum lactate levels between CTRL and W4 CPZ groups of CX₃CR1^GFP/GFP mice we used an unpaired t test (^#P < 0.05, ^##P < 0.01, ^###P < 0.001).

Acknowledgments

This work was supported by Grants NMSS_PP3395, Cal-BRAIN349087, and UCSF_RAP7500634, UCSF Department of Radiology seed Grants 14-04 and 14-05; NIH Grants R01CA172845 and R01NS102156; NIH Hyperpolarized MRI Technology Resource Center Grant P41EB013598; fellowships from the Flemish Institute for Science and Technology and National Multiple Sclerosis Society (FG-1507-05297); and Fondazione Italiana Sclerosi Multipla Senior Research Fellowship 2014/B/1 (to A.D.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1613345114/-/DCSupplemental.

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PERMALINK

Hyperpolarized 13C MR metabolic imaging can detect neuroinflammation in vivo in a multiple sclerosis murine model

Caroline Guglielmetti

Chloé Najac

Alessandro Didonna

Annemie Van der Linden

Sabrina M Ronen

Myriam M Chaumeil

Series information

Significance

Abstract

Results

Histological and MRI Characterization of the CPZ Mouse Model.

Fig. 1.

Increased Production of HP [1-13C]Lactate Following MP Activation in CPZ-Induced Lesions.

Fig. S1.

Fig. 2.

Fig. S2.

Fig. 3.

The Increase in HP [1-13C]Lactate Signal Is Associated with Up-Regulation of PDK1 in Activated MPs and Decreased PDH Activity in the Corpus Callosum.

Fig. 4.

Fig. S3.

HP [1-13C]Lactate Levels Are Not Increased in the Corpus Callosum of CX3CR1GFP/GFP Transgenic Mice Harboring Microglial Activation Deficiency, Suggesting a Central Role of MPs to the Detected HP [1-13C]Lactate Signal.

Fig. 5.

Fig. S4.