Raimo A. Salo

Comparison of seven different anesthesia protocols for nicotine pharmacologic magnetic resonance imaging in rat.

Pharmacologic MRI (phMRI) is a non-invasive in vivo imaging method, which can evaluate the drug effects on the brain and provide complementary information to ex vivo techniques. The preclinical phMRI studies usually require anesthesia to reduce the motion and stress of the animals. The anesthesia, however, is a crucial part of the experimental design, as it may modulate the neural drug-induced (de)activation and hemodynamic coupling. Therefore, the aim of the present study was to address this methodologic question by performing phMRI experiments with five anesthetics (α-chloralose, isoflurane, medetomidine, thiobutabarbital, and urethane) and seven anesthesia protocols. Nicotine, a widely studied psychostimulant, was administered to rats while measuring blood oxygenation level-dependent (BOLD) signals. Notably different responses were observed depending on the anesthetic used. The highest responses were measured in urethane-anesthetized rats whereas the responses were hardly noticeable in α-chloralose group. As urethane is not commonly used in phMRI, hemodynamic coupling under urethane anesthesia was investigated with functional cerebral blood flow (CBF) and volume-weighted (CBVw) imaging, and simultaneous electrophysiologic and BOLD measurements. The BOLD, CBF, and CBVw measurements in response to nicotine were highly correlated (R(2) ≥ 0.70, p<0.001). BOLD values correlated well (R(2)=0.43, p<10(-6)) with local field potential (LFP) spectral power (13-70Hz) during pharmacologic stimulation. These findings indicate that urethane anesthesia combined with BOLD contrast provides a robust protocol for nicotine phMRI studies. As urethane has mild effects to individual receptor systems, and coupling between electrophysiologic activity and hemodynamic response is maintained, this anesthetic may also be suitable for other phMRI studies.

Functional connectivity under six anesthesia protocols and the awake condition in rat brain

&NA; Resting‐state functional magnetic resonance imaging (rsfMRI) is a translational imaging method with great potential in several neurobiologic applications. Most preclinical rsfMRI studies are performed in anesthetized animals, but the confounding effects of anesthesia on the measured functional connectivity (FC) are poorly understood. Therefore, we measured FC under six commonly used anesthesia protocols and compared the findings with data obtained from awake rats. The results demonstrated that each anesthesia protocol uniquely modulated FC. Connectivity patterns obtained under propofol and urethane anesthesia were most similar to that observed in awake rats. FC patterns in the &agr;‐chloralose and isoflurane‐medetomidine combination groups had moderate to good correspondence with that in the awake group. The FC patterns in the isoflurane and medetomidine groups differed most from that in the awake rats. These results can be directly exploited in rsfMRI study designs to improve the data quality, comparability, and interpretation. HighlightsEach anesthesia protocol uniquely modulated functional connectivity (FC).FC obtained under propofol and urethane anesthesia was most similar to the awake rats.FC in &agr;‐chloralose and isoflurane‐medetomidine groups resembled well the awake group.FC in the isoflurane and medetomidine groups differed most from the awake rats.These results can be exploited in FC study designs and data interpretation.

Diffusion tensor MRI shows progressive changes in the hippocampus and dentate gyrus after status epilepticus in rat – histological validation with Fourier-based analysis

ABSTRACT Imaging markers for monitoring disease progression, recovery, and treatment efficacy are a major unmet need for many neurological diseases, including epilepsy. Recent evidence suggests that diffusion tensor imaging (DTI) provides high microstructural contrast even outside major white matter tracts. We hypothesized that in vivo DTI could detect progressive microstructural changes in the dentate gyrus and the hippocampal CA3bc in the rat brain after status epilepticus (SE). To test this hypothesis, we induced SE with systemic kainic acid or pilocarpine in adult male Wistar rats and subsequently scanned them using in vivo DTI at five time‐points: prior to SE, and 10, 20, 34, and 79 days post SE. In order to tie the DTI findings to changes in the tissue microstructure, myelin‐ and glial fibrillary acidic protein (GFAP)‐stained sections from the same animals underwent Fourier analysis. We compared the Fourier analysis parameters, anisotropy index and angle of myelinated axons or astrocyte processes, to corresponding DTI parameters, fractional anisotropy (FA) and the orientation angle of the principal eigenvector. We found progressive detectable changes in DTI parameters in both the dentate gyrus (FA, axial diffusivity [D||], linear anisotropy [CL] and spherical anisotropy [CS], p<0.001, linear mixed‐effects model [LMEM]) and the CA3bc (FA, D||, CS, and angle, p<0.001, LMEM; CL and planar anisotropy [CP], p<0.01, LMEM) post SE. The Fourier analysis revealed that both myelinated axons and astrocyte processes played a role in the water diffusion anisotropy changes detected by DTI in individual portions of the dentate gyrus (suprapyramidal blade, mid‐portion, and infrapyramidal blade). In the whole dentate gyrus, myelinated axons markedly contributed to the water diffusion changes. In CA3bc as well as in CA3b and CA3c, both myelinated axons and astrocyte processes contributed to water diffusion anisotropy and orientation. Our study revealed that DTI is a promising method for noninvasive detection of microstructural alterations in the hippocampus proper. These alterations may be potential imaging markers for epileptogenesis. HighlightsIn vivo DTI detected progressive changes in the DG and CA3bc after status epilepticus.Fourier analysis quantified changes in tissue metrics on histological sections.Contribution of myelinated axons and astrocyte processes to water diffusion changes.In vivo DTI, a promising method for noninvasive detection of microstructural changes.

Resting-state functional MRI as a tool for evaluating brain hemodynamic responsiveness to external stimuli in rats

Anesthesia is a major confounding factor in functional MRI (fMRI) experiments attributed to its effects on brain function. Recent evidence suggests that parameters obtained with resting‐state fMRI (rs‐fMRI) are coupled with anesthetic depth. Therefore, we investigated whether parameters obtained with rs‐fMRI, such as functional connectivity (FC), are also directly related to blood‐oxygen‐level–dependent (BOLD) responses.

Global Functional Connectivity Differences between Sleep-Like States in Urethane Anesthetized Rats Measured by fMRI

Sleep is essential for nervous system functioning and sleep disorders are associated with several neurodegenerative diseases. However, the macroscale connectivity changes in brain networking during different sleep states are poorly understood. One of the hindering factors is the difficulty to combine functional connectivity investigation methods with spontaneously sleeping animals, which prevents the use of numerous preclinical animal models. Recent studies, however, have implicated that urethane anesthesia can uniquely induce different sleep-like brain states, resembling rapid eye movement (REM) and non-REM (NREM) sleep, in rodents. Therefore, the aim of this study was to assess changes in global connectivity and topology between sleep-like states in urethane anesthetized rats, using blood oxygenation level dependent (BOLD) functional magnetic resonance imaging. We detected significant changes in corticocortical (increased in NREM-like state) and corticothalamic connectivity (increased in REM-like state). Additionally, in graph analysis the modularity, the measure of functional integration in the brain, was higher in NREM-like state than in REM-like state, indicating a decrease in arousal level, as in normal sleep. The fMRI findings were supported by the supplementary electrophysiological measurements. Taken together, our results show that macroscale functional connectivity changes between sleep states can be detected robustly with resting-state fMRI in urethane anesthetized rats. Our findings pave the way for studies in animal models of neurodegenerative diseases where sleep abnormalities are often one of the first markers for the disorder development.

Implantable RF-coil with multiple electrodes for long-term EEG-fMRI monitoring in rodents.

BACKGROUND Simultaneous EEG-fMRI is a valuable tool in the clinic as it provides excellent temporal and spatial information about normal and diseased brain function. In pre-clinical research with small rodents, obtaining simultaneous EEG-fMRI in longitudinal studies faces a number of challenges, including issues related to magnetic susceptibility artifacts. NEW METHOD Here, we demonstrate a method for permanent MRI RF-coil and EEG electrode implantation in rats that is suitable for long-term chronic follow-up studies in both stimulus and resting-state fMRI paradigms. RESULTS Our findings showed that the screw-free implantation method is well suited for long-term follow-up studies in both freely moving video-EEG settings and fMRI without causing MRI susceptibility artifacts. Furthermore, the results demonstrated that a multimodal approach can be used to track the progression of structural and functional changes. COMPARISON WITH EXISTING METHODS The quality of both MRI and EEG data were comparable to those obtained with traditional methods with the benefit of combining them into artifact-free simultaneous recordings. The signal-to-noise ratios of the MRI images obtained with the implanted RF-coil were similar to those using a quadrature coil and were therefore suitable for resting-state fMRI experiments. Similarly, EEG data collected with the RF-coil/electrode set-up were comparable to EEG recorded with traditional epidural screw electrodes. CONCLUSION This new multimodal EEG-fMRI approach provides a novel tool for concomitant analysis and follow-up of anatomic and functional MRI, as well as electrographic changes in a preclinical research.

Dose-response effect of acute phencyclidine on functional connectivity and dopamine levels, and their association with schizophrenia-like symptom classes in rat

ABSTRACT Current drug treatments for schizophrenia (SCZ) can alleviate positive symptoms, but have little effect on the negative symptoms and cognitive deficits that are difficult to translate into preclinical models for drug development. Therefore, we aimed to determine the dose‐response effects of acute phencyclidine (PCP, 1.0–5.0 mg/kg) on rat brain connectivity and detect markers for different SCZ‐like symptoms. Pharmacological functional magnetic resonance imaging (phMRI) and microdialysis were used to investigate PCP‐induced effects on functional connectivity (FC) and dopamine levels, respectively. Next, we evaluated the association between PCP‐induced changes in imaging parameters and behavior. PCP at doses of 3.0–5.0 mg/kg induced fMRI signal changes in several brain regions associated with SCZ. Additionally, the FC was globally disturbed, dopamine levels increased, and locomotor activity increased, reflecting the manifestation of SCZ‐like positive symptoms. A distinct pattern in the measures was observed at lower PCP doses (1.0–2.0 mg/kg); PCP induced fMRI signal changes in the fronto‐cortical regions, and increased dopamine levels in the medial prefrontal cortex. In addition to the dysconnectivity of these regions, the hippocampal FC was disrupted. These observations are consistent with the induction of SCZ‐like cognitive deficits and negative symptoms, which were observed as impaired novel object recognition and decreased social interaction. No indicators for positive symptoms were observed at lower PCP doses. We conclude that acute PCP induces SCZ‐like symptom classes in a dose‐dependent manner; PCP doses of 1.0–2.0 mg/kg are more suitable for modeling SCZ‐like negative symptoms and cognitive deficits, while SCZ‐like positive symptoms dominate at doses of 3.0–5.0 mg/kg. HIGHLIGHTSDifferent phencyclidine (PCP) doses induce distinct schizophrenia (SCZ)‐like symptoms.Functional connectivity (FC) is modulated dose‐dependently by PCP (1–5 mg/kg s.c.)At 3–5 mg/kg, PCP causes global disruption of FC and SCZ‐like positive symptoms.At 1–2 mg/kg, changes in FC indicate SCZ‐like negative and cognitive symptoms.Findings can facilitate the use of the acute PCP model in drug development for SCZ.

Quantification of anisotropy and orientation in 3D electron microscopy and diffusion tensor imaging in injured rat brain

&NA; Diffusion tensor imaging (DTI) reveals microstructural features of grey and white matter non‐invasively. The contrast produced by DTI, however, is not fully understood and requires further validation. We used serial block‐face scanning electron microscopy (SBEM) to acquire tissue metrics, i.e., anisotropy and orientation, using three‐dimensional Fourier transform‐based (3D‐FT) analysis, to correlate with fractional anisotropy and orientation in DTI. SBEM produces high‐resolution 3D data at the mesoscopic scale with good contrast of cellular membranes. We analysed selected samples from cingulum, corpus callosum, and perilesional cortex of sham‐operated and traumatic brain injury (TBI) rats. Principal orientations produced by DTI and 3D‐FT in all samples were in good agreement. Anisotropy values showed similar patterns of change in corresponding DTI and 3D‐FT parameters in sham‐operated and TBI rats. While DTI and 3D‐FT anisotropy values were similar in grey matter, 3D‐FT anisotropy values were consistently lower than fractional anisotropy values from DTI in white matter. We also evaluated the effect of resolution in 3D‐FT analysis. Despite small angular differences in grey matter samples, lower resolution datasets provided reliable results, allowing for analysis of larger fields of view. Overall, 3D SBEM allows for more sophisticated validation studies of diffusion imaging contrast from a tissue microstructural perspective. HighlightsSBEM produces mesoscale, high‐resolution 3D images of tissue microstructure.SBEM visualises cellular membranes, i.e. the barriers to the water diffusion.3D‐FT analysis on SBEM data extracts tissue metrics equivalent to DTI metrics.We extracted 3D‐FT and DTI metrics from white and grey matter of sham and TBI rats.The tissue metrics from the 3D‐FT in SBEM reflect changes in DTI metrics after TBI.

Awake Rat Brain Functional Magnetic Resonance Imaging Using Standard Radio Frequency Coils and a 3D Printed Restraint Kit

Functional magnetic resonance imaging (fMRI) is a powerful noninvasive tool for studying spontaneous resting state functional connectivity (RSFC) in laboratory animals. Brain function can be significantly affected by generally used anesthetics, however, rendering the need for awake imaging. Only a few different awake animal habituation protocols have been presented, and there is a critical need for practical and improved low-stress techniques. Here we demonstrate a novel restraint approach for awake rat RSFC studies. Our custom-made 3D printed restraint kit is compatible with a standard Bruker Biospin MRI rat bed, rat brain receiver coil, and volume transmitter coil. We also implemented a progressive habituation protocol aiming to minimize the stress experienced by the rats, and compared RSFC between awake, lightly sedated, and isoflurane-anesthetized rats. Our results demonstrated that the 3D printed restraint kit was suitable for RSFC studies of awake rats. During the short 4-day habituation period, the plasma corticosterone concentration, movement, and heart rate, which were measured as stress indicators, decreased significantly, indicating adaptation to the restraint protocol. Additionally, 10 days after the awake MRI session, rats exhibited no signs of depression or anxiety based on open-field and sucrose preference behavioral tests. The RSFC data revealed significant changes in the thalamo-cortical and cortico-cortical networks between the awake, lightly sedated, and anesthetized groups, emphasizing the need for awake imaging. The present work demonstrates the feasibility of our custom-made 3D printed restraint kit. Using this kit, we found that isoflurane markedly affected brain connectivity compared with that in awake rats, and that the effect was less pronounced, but still significant, when light isoflurane sedation was used instead.