In vivo functional brain mapping using ultra-high-field fMRI in awake common marmosets.

The common marmoset (Callithrix jacchus) is gaining attention in the field of cognitive neuroscience. The development of an effective protocol for fMRI data acquisition in awake marmosets is a key factor in developing reliable comparative studies. Here, we describe a protocol to obtain fMRI data in awake marmosets using auditory and visual stimulation. We describe steps for surgical and anesthesia procedures, MRI training, and positioning the marmosets within an MRI-compatible body restraint. We then detail fMRI scanning and preprocessing of functional images. For complete details on the use and execution of this protocol, please refer to Jafari et al. (2023).1.

Intracellular Acidification in a Rat C6 Glioma Model following Cariporide Injection Investigated by CEST-MRI.

Acidification of cancerous tissue induced pharmacologically may slow tumor growth and can be detected using magnetic resonance imaging. Numerous studies have shown that pharmacologically inhibiting specific transporters, such as the Na+/H+ exchanger 1 (NHE1), can alter glycolitic metabolism and affect tumor acidosis. The sodium proton exchanger inhibitor Cariporide can acidify U87MG gliomas in mice. This study aimed to determine whether Cariporide could acidify C6 glioma tumors in rats with an intact immune system. C6 glioma cells were implanted in the right brain hemisphere of ten rats. Chemical exchange saturation transfer (CEST) MRI (9.4T) was acquired on days 7-8 and 14-15 after implantation to measure in vivo tissue intracellular pH (pHi) within the tumors and on the contralateral side. pHi was basic relative to contralateral tissue at both time points assessed using the amine and amide concentration-independent detection (AACID) value. On day 14-15, measurements were made before and up to 160 min after Cariporide injection (N = 6). Twenty minutes after drug injection, the average AACID value in the tumor significantly increased by ∼6.4% compared to pre-injection, corresponding to 0.31 ± 0.20 lower pHi, while in contralateral tissue, AACID value increased significantly by ∼4.3% compared to pre-injection, corresponding to 0.22 ± 0.19 lower pHi. Control rats without tumors showed no changes following injection of Cariporide dissolved in 10% or 1% DMSO and diluted in PBS. This study demonstrates the sensitivity of CEST-based pH-weighted imaging for monitoring the response of tumors to pharmacologically induced acidification.

Glucose Infusion Induced Change in Intracellular pH and Its Relationship with Tumor Glycolysis in a C6 Rat Model of Glioblastoma.

INTRODUCTION: The reliance on glycolytic metabolism is a hallmark of tumor metabolism. Excess acid and protons are produced, leading to an acidic tumor environment. Therefore, we explored the relationship between the tumor glycolytic metabolism and tissue pH by comparing 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and hyperpolarized [1-13C]pyruvate MR spectroscopy imaging (MRSI) to chemical exchange saturation transfer (CEST) MRI measurements of tumor pH. METHODS: 106 C6 glioma cells were implanted in the brains of male Wistar rats (N = 11) using stereotactic surgery. A 60-min PET acquisition after a bolus of FDG was performed at 11-13 days post implantation, and standardized uptake value (SUV) was calculated. CEST measurements were acquired the following day before and during constant infusion of glucose solution. Tumor intracellular pH (pHi) was evaluated using amine and amide concentration-independent detection (AACID) CEST MRI. The change of pHi (∆pHi) was calculated as the difference between pHi pre- and during glucose infusion. Rats were imaged immediately with hyperpolarized [1-13C]pyruvate MRSI. Regional maps of the ratio of Lac:Pyr were acquired. The correlations between SUV, Lac:Pyr ratio, and ∆pHi were evaluated using Pearson's correlation. RESULTS: A decrease of 0.14 in pHi was found after glucose infusion in tumor region. Significant correlations between tumor glycolysis measurements of Lac:Pyr and ∆pHi within the tumor (ρ = 0.83, P = 0.01) and peritumoral region (ρ = 0.76, P = 0.028) were observed. No significant correlations were found between tumor SUV and ∆pHi within the tumor (ρ = - 0.45, P = 0.17) and peritumor regions (ρ = - 0.6, P = 0.051). CONCLUSION: AACID detected the changes in pHi induced by glucose infusion. Significant correlations between tumor glycolytic measurement of Lac:Pyr and tumoral and peritumoral pHi and ∆pHi suggest the intrinsic relationship between tumor glycolytic metabolism and the tumor pH environment as well as the peritumor pH environment.

Frontoparietal connectivity as a product of convergent evolution in rodents and primates: functional connectivity topologies in grey squirrels, rats, and marmosets.

Robust frontoparietal connectivity is a defining feature of primate cortical organization. Whether mammals outside the primate order, such as rodents, possess similar frontoparietal functional connectivity organization is a controversial topic. Previous work has primarily focused on comparing mice and rats to primates. However, as these rodents are nocturnal and terrestrial, they rely much less on visual input than primates. Here, we investigated the functional cortical organization of grey squirrels which are diurnal and arboreal, thereby better resembling primate ecology. We used ultra-high field resting-state fMRI data to compute and compare the functional connectivity patterns of frontal regions in grey squirrels (Sciurus carolinensis), rats (Rattus norvegicus), and marmosets (Callithrix jacchus). We utilized a fingerprinting analysis to compare interareal patterns of functional connectivity from seeds across frontal cortex in all three species. The results show that grey squirrels, but not rats, possess a frontoparietal connectivity organization that resembles the connectivity pattern of marmoset lateral prefrontal cortical areas. Since grey squirrels and marmosets have acquired an arboreal way of life but show no common arboreal ancestor, the expansion of the visual system and the formation of a frontoparietal connectivity architecture might reflect convergent evolution driven by similar ecological niches in primates and tree squirrels.

Effects of Memantine and High Dose Vitamin D on Gait in Male APP/PS1 Alzheimer's Disease Mice Following Vitamin D Deprivation.

BACKGROUND: Altered gait is a frequent feature of Alzheimer's disease (AD), as is vitamin D deficiency. Treatment with memantine and vitamin D can protect cortical axons from exposure to amyloid-ß and glutamate toxicity, suggesting this combination may mitigate altered gait in AD. OBJECTIVE: Investigate the effects of vitamin D deprivation and subsequent treatment with memantine and vitamin D enrichment on gait performance in APPswe/PS1dE9 mice. METHODS: Male APPswe/PS1dE9 mice were split into four groups (n = 14 each) at 2.5 months of age. A control group was fed a standard diet throughout while the other three groups started a vitamin D-deficient diet at month 6. One group remained on this deficient diet for the rest of the study. At month 9, the other two groups began treatment with either memantine alone or memantine combined with 10 IU/g of vitamin D. Gait was assessed using CatWalk at months 6, 9, 12, and 15. RESULTS: Vitamin D deprivation led to a 13% increase in hind stride width by month 15 (p < 0.001). Examination of the treatment groups at month 15 revealed that mice treated with memantine alone still showed an increase in hind stride width compared to controls (p < 0.01), while mice treated with memantine and vitamin D did not (p = 0.21). CONCLUSION: Vitamin D deprivation led to impaired postural control in the APPswe/PS1dE9 model. Treatment with memantine and vitamin D, but not memantine alone, prevented this impairment. Future work should explore the potential for treatments incorporating vitamin D supplementation to improve gait in people with AD.

Effect of Memantine Treatment and Combination with Vitamin D Supplementation on Body Composition in the APP/PS1 Mouse Model of Alzheimer's Disease Following Chronic Vitamin D Deficiency.

BACKGROUND: Vitamin D deficiency and altered body composition are common in Alzheimer's disease (AD). Memantine with vitamin D supplementation can protect cortical axons against amyloid-ß exposure and glutamate toxicity. OBJECTIVE: To study the effects of vitamin D deprivation and subsequent treatment with memantine and vitamin D enrichment on whole-body composition using a mouse model of AD. METHODS: Male APPswe/PS1dE9 mice were divided into four groups at 2.5 months of age: the control group (n = 14) was fed a standard diet throughout; the remaining mice were started on a vitamin D-deficient diet at month 6. The vitamin D-deficient group (n = 14) remained on the vitamin D-deficient diet for the rest of the study. Of the remaining two groups, one had memantine (n = 14), while the other had both memantine and 10 IU/g vitamin D (n = 14), added to their diet at month 9. Serum 25(OH)D levels measured at months 6, 9, 12, and 15 confirmed vitamin D levels were lower in mice on vitamin D-deficient diets and higher in the vitamin D-supplemented mice. Micro-computed tomography was performed at month 15 to determine whole-body composition. RESULTS: In mice deprived of vitamin D, memantine increased bone mineral content (8.7% increase, p < 0.01) and absolute skeletal tissue mass (9.3% increase, p < 0.05) and volume (9.2% increase, p < 0.05) relative to controls. This was not observed when memantine treatment was combined with vitamin D enrichment. CONCLUSION: Combination treatment of vitamin D and memantine had no negative effects on body composition. Future studies should clarify whether vitamin D status impacts the effects of memantine treatment on bone physiology in people with AD.

Topiramate induces acute intracellular acidification in glioblastoma.

Reversal of the intracellular/extracellular pH gradient is a hallmark of malignant tumors and is an important consideration in evaluating tumor growth potential and the effectiveness of anticancer therapies. Glioblastoma multiforme (GBM) brain tumors have increased expression of the carbonic anhydrase (CA) isozymes CAII, CAIX and CAXII that contribute to the altered regulation of intracellular pH (pHi). The anti-epileptic drug topiramate (TPM) inhibits CA action and may acidify the tumor intracellular compartment. In-vivo detection of acute tumor acidification could aid in cancer diagnosis and monitoring treatment response. Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) has been used to measure tissue pH. Using a recently developed CEST-MRI method called amine/amide concentration independent detection (AACID), we have previously shown intracellular acidification caused by single dose of lonidamine. The current study aims to evaluate the intracellular acidification induced by a single dose of the clinically approved drug TPM. Brain tumors were induced in NU/NU mice by injecting 105 U87 human glioblastoma multiforme cells into the right frontal lobe. Using a 9.4T MRI scanner AACID measurements were acquired, before and after administration of TPM (dose: 120 mg/kg, intraperitoneal), 15 ± 2 days after tumor cell implantation. TPM administration induced acute intracellular acidification (average ± SD: baseline AACID = 1.14 ± 0.05; post AACID = 1.19 ± 0.05, paired ttest p = 0.02) in implanted brain tumors. In contrast, contralateral tissue showed no change in AACID value. These results suggest that topiramate can rapidly induce a tumor specific physiological change detectable by AACID CEST. This pH challenge paradigm could be exploited to aid in tumor detection and monitoring treatment response.

Quantitative tissue pH measurement during cerebral ischemia using amine and amide concentration-independent detection (AACID) with MRI.

Tissue pH is an indicator of altered cellular metabolism in diseases including stroke and cancer. Ischemic tissue often becomes acidic due to increased anaerobic respiration leading to irreversible cellular damage. Chemical exchange saturation transfer (CEST) effects can be used to generate pH-weighted magnetic resonance imaging (MRI) contrast, which has been used to delineate the ischemic penumbra after ischemic stroke. In the current study, a novel MRI ratiometric technique is presented to measure absolute pH using the ratio of CEST-mediated contrast from amine and amide protons: amine/amide concentration-independent detection (AACID). Effects of CEST were observed at 2.75 parts per million (p.p.m.) for amine protons and at 3.50 p.p.m. for amide protons downfield (i.e., higher frequency) from bulk water. Using numerical simulations and in vitro MRI experiments, we showed that pH measured using AACID was independent of tissue relaxation time constants, macromolecular magnetization transfer effects, protein concentration, and temperature within the physiologic range. After in vivo pH calibration using phosphorus ((31)P) magnetic resonance spectroscopy ((31)P-MRS), local acidosis is detected in mouse brain after focal permanent middle cerebral artery occlusion. In summary, our results suggest that AACID represents a noninvasive method to directly measure the spatial distribution of absolute pH in vivo using CEST MRI.

Comparison of T2 and LASER T2dagger image contrast in a rat model of acute cerebral ischemia.

Previous studies have shown that T2(dagger)-weighted magnetic resonance images acquired using localization by adiabatic selective refocusing (LASER) can provide early tissue contrast following ischemia, possibly due to alterations in microscopic susceptibility within the tissue. The purpose of this study was to make a direct in vivo comparison of T2-, T2(dagger)- and diffusion-weighted image contrast during acute ischemia. Acute middle cerebral artery (MCA) occlusion was attempted in 14 rats using a modified Tamura approach incorporating electrocoagulation of the left MCA. T2(dagger)-weighted LASER images (Echo Time [TE]=108 ms), T2-weighted Carr-Purcell-Meiboom-Gill (CPMG) images (TE=110 ms) and diffusion-weighted images (b value=105 s/mm(2)) were acquired at 4 T within 1.5 h of ischemia onset. Tissue contrast in the MCA territory was quantified for histologically verified ischemic tissue (n=6) and in sham controls (n=4). T2(dagger)-weighted LASER images demonstrated greater contrast compared to the T2-weighted CPMG images, and more focal contrast compared to the diffusion-weighted images, suggesting different contrast mechanisms were involved.