Excised Brain Tissue Research Articles

Ultrasound-Guided Mechanical High-Intensity Focused Ultrasound (Histotripsy) Through an Acoustically Permeable Polyolefin-Based Cranioplasty Device.

Recently, a biocompatible, polyolefin-based cranioplasty device was developed to allow ultrasound (US) transmission into the intracranial space with minimal distortion. In this study, we investigated the in vitro feasibility of applying US-guided histotripsy procedures across the prosthesis. Pressure waveforms and beam profiles were collected for single- and multi-element histotripsy transducers. Then, high-speed optical images of the bubble cloud with and without the prosthesis were collected in water and tissue-mimicking agarose gel phantoms. Finally, red blood cell (RBC) tissue phantom and excised brain tissue experiments were completed to test the ablative efficacy across the prosthesis. Single element tests revealed increased pressure loss with increasing transducer frequency and increasing transducer-to-prosthesis angle. Array transducer measurements at 1 MHz showed average pressure losses of >50% across the prosthesis. Aberration correction recovered up to 18% of the pressure lost, and high-speed optical imaging in water, agarose gels, and RBC phantoms demonstrated that histotripsy bubble clouds could be generated across the prosthesis at pulse repetition frequencies of 50-500 Hz. Histologic analysis revealed a complete breakdown of brain tissue treated across the prosthesis. Conclusion & Significance: Overall, the results of this study demonstrate that the cranial prosthesis may be used as an acoustic window through which intracranial histotripsy can be applied under US guidance without the need for large transcranial array transducers.

Experimentally unsupervised deconvolution for light-sheet microscopy with propagation-invariant beams

Deconvolution is a challenging inverse problem, particularly in techniques that employ complex engineered point-spread functions, such as microscopy with propagation-invariant beams. Here, we present a deep-learning method for deconvolution that, in lieu of end-to-end training with ground truths, is trained using known physics of the imaging system. Specifically, we train a generative adversarial network with images generated with the known point-spread function of the system, and combine this with unpaired experimental data that preserve perceptual content. Our method rapidly and robustly deconvolves and super-resolves microscopy images, demonstrating a two-fold improvement in image contrast to conventional deconvolution methods. In contrast to common end-to-end networks that often require 1000–10,000s paired images, our method is experimentally unsupervised and can be trained solely on a few hundred regions of interest. We demonstrate its performance on light-sheet microscopy with propagation-invariant Airy beams in oocytes, preimplantation embryos and excised brain tissue, as well as illustrate its utility for Bessel-beam LSM. This method aims to democratise learned methods for deconvolution, as it does not require data acquisition outwith the conventional imaging protocol.

Fast intraoperative histology-based diagnosis of gliomas with third harmonic generation microscopy and deep learning

Management of gliomas requires an invasive treatment strategy, including extensive surgical resection. The objective of the neurosurgeon is to maximize tumor removal while preserving healthy brain tissue. However, the lack of a clear tumor boundary hampers the neurosurgeon’s ability to accurately detect and resect infiltrating tumor tissue. Nonlinear multiphoton microscopy, in particular higher harmonic generation, enables label-free imaging of excised brain tissue, revealing histological hallmarks within seconds. Here, we demonstrate a real-time deep learning-based pipeline for automated glioma image analysis, matching video-rate image acquisition. We used a custom noise detection scheme, and a fully-convolutional classification network, to achieve on average 79% binary accuracy, 0.77 AUC and 0.83 mean average precision compared to the consensus of three pathologists, on a preliminary dataset. We conclude that the combination of real-time imaging and image analysis shows great potential for intraoperative assessment of brain tissue during tumor surgery.

Distinctive ionic transport of freshly excised human epileptogenic brain tissue.

Epileptogenic lesions have higher concentrations of sodium than does normal brain tissue. Such lesions are palpably recognized by a surgeon and then excised in order to eliminate epileptic seizures with their associated abnormal electrical behavior. Here, we study the frequency-dependent electrical conductivities of lesion-laden tissues excised from the brains of epilepsy patients. The low-frequency (<1000 Hz) conductivity of biological tissue primarily probes extracellular solvated sodium-cations traveling parallel to membranes within regions bounded by blockages. This conductivity rises monotonically toward saturation as the frequency surpasses the rate with which diffusing solvated sodium cations encounter blockages. We find that saturation occurs at dramatically higher frequencies in excised brain tissue containing epileptogenic lesions than it does in normal brain tissue. By contrast, such an effect is not reported for tumors embedded in other excised biological tissue. All told, epileptogenic lesions generate frequency-dependent conductivities that differ qualitatively from those of both normal brain tissues and tumors.

Thermoacoustic tomography of germinal matrix hemorrhage in neonatal mouse cerebrum.

Microwave-induced thermoacoustic tomography (TAT) has potential for detecting germinal matrix hemorrhage (GMH). However, it has not been demonstrated in vivo. To demonstrate the feasibility of TAT for in vivo detecting GMH by using neonatal mouse. A cylindrical-scanning TAT system was developed with optimized microwave irradiation and ultrasound detection for neonatal mouse imaging. Neonatal mice were used to develop GMH model by injection of autologous blood into the periventricular region. After TAT experiments, the animals were sacrificed, frozen and excised to validate the TAT findings. The detailed comparative analyses of the TAT images and corresponding photographs of the excised brain tissues were conducted. Satisfactory matches are identified between the TAT images and corresponding histological sections, in terms of the shape and size of the brain tissues. Some organs and tissues were also identified. Particularly, comparing to the corresponding histological sections, using TAT enables to more accurately detect the hematoma region at different depths in the neonatal mouse brain. This study demonstrates for the first time that TAT can detect GMH in neonatal mouse cerebrum in vivo. This represents the first important step towards the in vivo diagnosis and grading of hemorrhage in the infant human brain.

A tissue chamber chip for assessing nanoparticle mobility in the extravascular space.

Although a plethora of nanoparticle configurations have been proposed over the past 10years, the uniform and deep penetration of systemically injected nanomedicines into the diseased tissue stays as a major biological barrier. Here, a 'Tissue Chamber' chip is designed and fabricated to study the extravascular transport of small molecules and nanoparticles. The chamber comprises a collagen slab, deposited within a PDMS mold, and an 800μm channel for the injection of the working solution. Through fluorescent microscopy, the dynamics of molecules and nanoparticles was estimated within the gel, under different operating conditions. Diffusion coefficients were derived from the analysis of the particle mean square displacements (MSD). For validating the experimental apparatus and the protocol for data analysis, the diffusion D of FITC-Dextran molecules of 4, 40 and 250kDa was first quantified. As expected, D reduces with the molecular weight of the dextran molecules. The MSD-derived diffusion coefficients were in good agreement with values derived via fluorescence recovery after photobleaching (FRAP), an alternative technique that solely applies to small molecules. Then, the transport of six nanoparticles with similar hydrodynamic diameters (~ 200nm) and different surface chemistries was quantified. Surface PEGylation was confirmed to favor the diffusion of nanoparticles within the collagen slab, whereas the surface decoration with hyaluronic acid (HA) chains reduced nanoparticle mobility in a way proportional to the HA molecular weight. To assess further the generality of the proposed approach, the diffusion of the six nanoparticles was also tested in freshly excised brain tissue slices. In these ex vivo experiments, the diffusion coefficients were 5-orders of magnitude smaller than for the Tissue Chamber chip. This was mostly ascribed to the lack of a cellular component in the chip. However, the trends documented for PEGylated and HA-coated nanoparticles in vitro were also confirmed ex vivo. This work demonstrates that the Tissue Chamber chip can be employed to effectively and efficiently test the extravascular transport of nanomedicines while minimizing the use of animals.

Germline and somatic mutations in STXBP1 with diverse neurodevelopmental phenotypes.

Objective:To expand the clinical phenotype associated with STXBP1 gene mutations and to understand the effect of STXBP1 mutations in the pathogenesis of focal cortical dysplasia (FCD).Methods:Patients with STXBP1 mutations were identified in various ways: as part of a retrospective cohort study of epileptic encephalopathy; through clinical referrals of individuals (10,619) with developmental delay (DD) for chromosomal microarray; and from a collection of 5,205 individuals with autism spectrum disorder (ASD) examined by whole-genome sequencing.Results:Seven patients with heterozygous de novo mutations affecting the coding region of STXBP1 were newly identified. Three cases had radiologic evidence suggestive of FCD. One male patient with early infantile epileptic encephalopathy, DD, and ASD achieved complete seizure remission following resection of dysplastic brain tissue. Examination of excised brain tissue identified mosaicism for STXBP1, providing evidence for a somatic mechanism. Cell-type expression analysis suggested neuron-specific expression. A comprehensive analysis of the published data revealed that 3.1% of severe epilepsy cases carry a pathogenic de novo mutation within STXBP1. By contrast, ASD was rarely associated with mutations in this gene in our large cohorts.Conclusions:STXBP1 mutations are an important cause of epilepsy and are also rarely associated with ASD. In a case with histologically proven FCD, an STXBP1 somatic mutation was identified, suggesting a role in its etiology. Removing such tissue may be curative for STXBP1-related epilepsy.

Measuring the local electrical conductivity of human brain tissue

The electrical conductivities of freshly excised brain tissues from 24 patients were measured. The diffusion-MRI of the hydrogen nuclei of water molecules from regions that were subsequently excised was also measured. Analysis of these measurements indicates that differences between samples' conductivities are primarily due to differences of their densities of solvated sodium cations. Concomitantly, the sample-to-sample variations of their diffusion constants are relatively small. This finding suggests that non-invasive in-vivo measurements of brain tissues' local sodium-cation density can be utilized to estimate its local electrical conductivity.

Intraoperative Spectroscopy with Ultrahigh Sensitivity for Image-Guided Surgery of Malignant Brain Tumors.

Intraoperative cancer imaging and fluorescence-guided surgery have attracted considerable interest because fluorescence signals can provide real-time guidance to assist a surgeon in differentiating cancerous and normal tissues. Recent advances have led to the clinical use of a natural fluorophore called protoporphyrin IX (PpIX) for image-guided surgical resection of high-grade brain tumors (glioblastomas). However, traditional fluorescence imaging methods have only limited detection sensitivity and identification accuracy and are unable to detect low-grade or diffuse infiltrating gliomas (DIGs). Here we report a low-cost hand-held spectroscopic device that is capable of ultrasensitive detection of protoporphyrin IX fluorescence in vivo, together with intraoperative spectroscopic data obtained from both animal xenografts and human brain tumor specimens. The results indicate that intraoperative spectroscopy is at least 3 orders of magnitude more sensitive than the current surgical microscopes, allowing ultrasensitive detection of as few as 1000 tumor cells. For detection specificity, intraoperative spectroscopy allows the differentiation of brain tumor cells from normal brain cells with a contrast signal ratio over 100. In vivo animal studies reveal that protoporphyrin IX fluorescence is strongly correlated with both MRI and histological staining, confirming that the fluorescence signals are highly specific to tumor cells. Furthermore, ex vivo spectroscopic studies of excised brain tissues demonstrate that the hand-held spectroscopic device is capable of detecting diffuse tumor margins with low fluorescence contrast that are not detectable with current systems in the operating room. These results open new opportunities for intraoperative detection and fluorescence-guided resection of microscopic and low-grade glioma brain tumors with invasive or diffusive margins.

Demeclocycline as a contrast agent for detecting brain neoplasms using confocal microscopy

Complete resection of brain tumors improves life expectancy and quality. Thus, there is a strong need for high-resolution detection and microscopically controlled removal of brain neoplasms. The goal of this study was to test demeclocycline as a contrast enhancer for the intraoperative detection of brain tumors. We have imaged benign and cancerous brain tumors using multimodal confocal microscopy. The tumors investigated included pituitary adenoma, meningiomas, glioblastomas, and metastatic brain cancers. Freshly excised brain tissues were stained in 0.75 mg ml−1 aqueous solution of demeclocyline. Reflectance images were acquired at 402 nm. Fluorescence signals were excited at 402 nm and registered between 500 and 540 nm. After imaging, histological sections were processed from the imaged specimens and compared to the optical images. Fluorescence images highlighted normal and cancerous brain cells, while reflectance images emphasized the morphology of connective tissue. The optical and histological images were in accordance with each other for all types of tumors investigated. Demeclocyline shows promise as a contrast agent for intraoperative detection of brain tumors.

Hyperspectral imaging signatures detect amyloidopathy in Alzheimer's mouse retina well before onset of cognitive decline.

Amyloidopathic disorders such as Alzheimer's disease present symptomology years after the entrenchment of amyloidogenic imbalance. The pathologic α-helix → β-strand conversion of amyloid β(1-42) and amyloid β(1-40) peptides causes neuronal death in the vicinity. Symptomology often presents only after significant neurodegeneration. This thus warrants early detection of amyloidopathy in Alzheimer's disease. Nonexistent modalities for direct identification and quantitation of soluble amyloid aggregates or (proto)fibrils forced us to undertake the development of a spectrophotometric technique to support ongoing drug design. Key requirements were independence from the need for extraneous staining, unambiguous amyloid aggregate detection, and minimal influence of interpretative errors. A Cytoviva instrument pivotal to this study captures scattering of light of visible-near-infrared (VNIR, 400-1000 nm) wavelengths within each pixel of the microscopic view field. We thus assembled a scattering intensity pattern database that provided "signatures" of amyloid aggregates. Comparison of unknown samples against this database enabled direct detection of amyloid aggregates. The technique was found useful for monitoring retinal and brain amyloidopathy in an ongoing preclinical anti-AD study, attesting to the technique's sensitivity and specificity. Interestingly, the technique was found applicable not just to excised brain tissue but also to isolated mouse retina. With the retina being heralded widely as a (diagnostic) extension of the CNS and retinal amyloidopathy occurring well before that in the brain, this development raises a possibility for the first direct retinal imaging diagnosis of early asymptomatic Alzheimer's disease.

Study of freshly excised brain tissues using terahertz imaging.

We demonstrated that tumors in freshly excised whole brain tissue could be differentiated clearly from normal brain tissue using a reflection-type terahertz (THz) imaging system. THz binary images of brain tissues with tumors indicated that the tumor boundaries in the THz images corresponded well to those in visible images. Grey and white-matter regions were distinguishable owing to the different distribution of myelin in the brain tissue. THz images corresponded closely with magnetic resonance imaging (MRI) results. The MRI and hematoxylin and eosin-stained microscopic images were investigated to account for the intensity differences in the THz images for fresh and paraffin-embedded brain tissue. Our results indicated that the THz signals corresponded to the cell density when water was removed. Thus, THz imaging could be used as a tool for label-free and real-time imaging of brain tumors, which would be helpful for physicians to determine tumor margins during brain surgery.

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)–Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)–biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p<0.001) in MR signal intensity or a significant decrease (p<0.001) in T1 relaxation, measured as %T1 change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p<0.0001) in T1 relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin–Gd-DTPA–biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p<0.001) and 3-nitrotyrosine (3-NT) (p<0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.

Plausible role of naringenin against cerebrally implanted C6 glioma cells in rats

Gliomas encompass a significant percentage of intrinsic neoplasms of the central nervous system in both adults and children. The constitutive activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B is the hallmark of glioma. The up-regulated protein kinase B could influence the expression of cyclooxygenase-2, an indicator of aggressive glioma. The present study was embarked to demonstrate the effect of naringenin (50 mg/kg bw for 30 days administrated orally) on PI3K, protein kinase B, and cyclooxygenase-2 in cerebrally implanted rat C6 glioma model. After the experimental period of 30 days, the animals were sacrificed and excised brain tissues were subjected to study the expressions of PI3K, protein kinase B, and cyclooxygenase-2 by reverse transcriptase polymerase chain reaction followed Western blot analysis. The activity of COX-2 (production of prostaglandin-E(2)) was also determined by high pressure liquid chromatography. The results showed that the naringenin could down-regulate the expressions of PI3K and protein kinase B along with activity and expression of cyclooxygenase-2 in C6 glioma cells implanted rat brain. In conclusion, it can be argued that the reduced expressions of phosphatidylinositol 3-kinase and protein kinase B in naringenin-treated glioma-induced rat brain might be involved in the down-regulation of cyclooxygenase-2 expression and activity. Thus, fine-tuned investigation of which will be helpful for targeted drug discovery against glioma.

Imaging of the muscarinic acetylcholine neuroreceptor in rats with the M2 selective agonist [18F]FP-TZTP

[(18)F]FP-TZTP is an M2 muscarinic subtype selective receptor-binding radiotracer used in vivo to image human and nonhuman primate brain following both bolus injection and infusion. In order to carry out repeated studies in rodents, the techniques developed for primates must be transferred to rodents with the same precision. This includes obtaining a metabolite-corrected input function. We compared bolus injection with constant infusion in rats that were awake or under isoflurane anesthesia. Brain-plasma and brain-blood distribution ratios were calculated by dividing brain (18)F concentrations, determined in vivo by positron emission tomography imaging with the Advanced Technology Laboratory Animal Scanner, ex vivo by direct counting in excised brain tissue or by quantitative autoradiography by the plasma or whole blood concentrations that had been corrected for metabolite contents. Blood volume constraints prevented adequate blood sampling to define a precise input function after bolus injection, thus preventing full kinetic analysis. Constant infusion, however, required fewer blood samples to define the input function, allowing calculation of distribution ratios, but complete equilibrium between plasma and tissue had not yet been reached after 120 min. Our results showed that the blood clearance and metabolism were too rapid to obtain a reproducible input function after bolus injection. The equilibrium distribution ratios did not lead to precise biochemical parameters, but the constant infusion was more suitable in that distribution ratios between tissue and plasma were statistically more precise. Constant infusion is the better approach for studying [(18)F]FP-TZTP by small animal imaging.

Focused ultrasound modulates region-specific brain activity

We demonstrated the in vivo feasibility of using focused ultrasound (FUS) to transiently modulate (through either stimulation or suppression) the function of regional brain tissue in rabbits. FUS was delivered in a train of pulses at low acoustic energy, far below the cavitation threshold, to the animal's somatomotor and visual areas, as guided by anatomical and functional information from magnetic resonance imaging (MRI). The temporary alterations in the brain function affected by the sonication were characterized by both electrophysiological recordings and functional brain mapping achieved through the use of functional MRI (fMRI). The modulatory effects were bimodal, whereby the brain activity could either be stimulated or selectively suppressed. Histological analysis of the excised brain tissue after the sonication demonstrated that the FUS did not elicit any tissue damages. Unlike transcranial magnetic stimulation, FUS can be applied to deep structures in the brain with greater spatial precision. Transient modulation of brain function using image-guided and anatomically-targeted FUS would enable the investigation of functional connectivity between brain regions and will eventually lead to a better understanding of localized brain functions. It is anticipated that the use of this technology will have an impact on brain research and may offer novel therapeutic interventions in various neurological conditions and psychiatric disorders.

Non-Mono-Exponential Attenuation of Water andN-Acetyl Aspartate Signals Due to Diffusion in Brain Tissue

Diffusion measurements were performed on water andN-acetyl aspartate (NAA) molecules in excised brain tissue using a wide range ofb-values (up to 28.3 × 106and 35.8 × 106s cm−2for water and NAA, respectively). The attenuation of the signals of water and NAA due to diffusion was measured at fixed diffusion times (tD). These measurements, in which the echo time (TE) was set to 70 ms, were repeated for several diffusion times ranging from 35 to 305 ms. Signal attenuations were fitted to mono-, bi-, and triexponential functions to obtain the apparent diffusion coefficients (ADCs) of these molecules at each diffusion time. From these experiments the following observations and conclusions were made: (1) Signal attenuation of water and NAA due to diffusion over the entire range ofbvalues examined is not monoexponential and the extracted ADCs depend on the diffusion time; (2) In the case of water the experimental data are best fitted by a triexponential function, while forbvalues up to 1 × 106s cm−2, a biexponential function seems to reproduce the experimental data as well as the triexponential function; (3) If only the low range ofbvalues are fitted (up to 0.5 × 106s cm−2) signal attenuation of water is monoexponential and insensitive totD; (4) Water ADCs decreased with the increase intDbut the relative population of the fast diffusing component increases such that at atDof 305 ms there is nearly a single population; (5) The major fast diffusion component of the water shows only very limited restriction; (6) NAA signal attenuation is biexponential and analysis of the lowb-value range gives only monoexponential decay, but the obtained ADC is sensitive to the diffusion time; (7) The ADCs obtained from fitting the data with a biexponential function decrease as diffusion time increases; (8) The relative population of the slow-diffusing component decreases with increasingtD; (9) Both the fast and the slow diffusing components of NAA show a considerable restriction by what seems to be a nonpermeable barrier from which two compartments, one of 7–8 μm and one of ∼1 μm, were calculated using the Einstein equation. It is suggested that the two compartments represent the NAA in cell bodies and in the intra-axonal space. The effect of the range of thebvalue used in the diffusion experiments on the results is discussed and used to reconcile some of the apparent discrepancies obtained in different experiments concerning water diffusion in brain tissue. The potential of NAA diffusion experiments to probe cellular structure is discussed.

Detection of Different Water Populations in Brain Tissue Using2H Single- and Double-Quantum-Filtered Diffusion NMR Spectroscopy

This paper presents the first simultaneous2H single- and double-quantum (SQ and DQ, respectively) diffusion study of excised brain tissue. The apparent diffusion coefficients (ADCs) of the2H SQ and DQ signals were measured at a fixed diffusion time (Δ − δ/3 = 21.3 ms) and as a function of the diffusion time to assess restricted diffusion [(Δ − δ/3) was changed from 21.3 to 271.3 ms]. As expected, the ADC of the SQ signal was higher than that of the DQ signal [0.53 ± 0.03 × 10−5(n= 3) and 0.30 ± 0.03 × 10−5cm2s−1(n= 4), respectively]. When the ADCs of the SQ and DQ signals were measured as a function of the diffusion time, two components, a fast and a slow component, were observed in each case. The ADCs for the SQ signal were 1.16 ± 0.2 × 10−5and 0.35 ± 0.06 × 10−5cm2s−1(n= 3) for the fast and the slow components, respectively. The ADCs for the DQ signal were 0.31 ± 0.05 × 10−5and ∼0.03 ± 0.03 × 10−5cm2s−1(n= 2) with the slow component being relatively small. Interestingly, the slow-diffusion component of the SQ signal was found to have an ADC similar to that of the fast component of the DQ signal. These results suggest that brain water can be divided into at least three water populations and that the DQ signal originates from water molecules which interact with slow-diffusing structural components of the brain. The new insights that one can obtain using simultaneous SQ and DQ diffusion measurement and the ability to distinguish among water populations in biological tissues using the above approach are discussed.

Application of continuous relaxation time distributions to the fitting of data from model systmes and excised tissue

Biological systems exhibit heterogeneity at many different levels, leading to the expectation of multiple relaxation time components for water protons in tissue samples. Traditional methods which fit the relaxation data to an a priori number of discrete components are open to observer bias in their interpretation of this data, and moreover, are intuitively less realistic for heterogeneous systems than methods which produce continuous relaxation time distributions. Previous validations of continuous distribution techniques have been made on simulated data assuming uniform Gaussian noise. In the current work we have investigated the ability of one particular linear inverse theory technique to reproduce known relaxation time distributions from the data on a controllable model system. Furthermore, using the experience gained on the model system, we have applied this same technique to the analysis of in vitro relaxation time measurements on excised brain tissue and found for water protons in white matter, four reproducible components for the transverse relaxation, whereas gray matter gave rise to only two. The longitudinal relaxation displayed only one component in either white matter or gray matter.

Assignment of resonances in the 1H spectrum of rat brain by two‐dimensional shift correlated and j‐resolved NMR spectroscopy

Two-dimensional shift-correlated (COSY) and J-resolved NMR spectroscopy was used to identify and assign resonance in the aliphatic region (0.8 to 4.5 ppm) of the 1H spectrum of acid extracts and tissue of rat brain. The chemical shift and spin-spin coupling constants of several resonances, which could not be resolved in one-dimensional spectra of tissue, were determined. These properties, together with the appropriate multiplet structure and scalar coupling patterns observed in 2D J-resolved and COSY spectra, were used to assign the resonances of lactate, threonine, alanine gamma-amino butyrate, N-acetyl aspartate, aspartate, glutamate, glutamine, taurine, and myo-inositol. The single peak observed at 1.33 ppm in the 1D 1H spectrum of excised brain tissue, which is generally assigned to lactate, was shown instead to consist of two overlapping peaks in 2D COSY contour plots. The intensity of both resonances increased considerably after death. The second resonance was assigned to threonine and the assignment was confirmed by ion-exchange chromatography. Several cross peaks were identified in 2D COSY spectra of intact brain tissue that were not present in similar spectra of the acid extract. The nonmetabolite resonances were assigned to macromolecules. Analysis of 2D COSY contour plots in conjunction with 1D spectra revealed that the total creatinine (creatinine + phosphocreatine) resonance at 3.0 ppm was overlapped by both GABA and macromolecules. This macromolecule resonance was present in a 2D COSY spectrum of the rat brain obtained in situ with a surface coil following cardiac arrest, indicating that its presence in the 2D spectrum of excised brain tissue did not arise from tissue disruption.