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Keywords:

  • epilepsy;
  • flavoprotein fluorescence;
  • human;
  • optical imaging;
  • pathophysiology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Flavoprotein Fluorescence Imaging in Vitro
  5. Flavoprotein Fluorescence Imaging of Human Brain Slices
  6. Analysis of Spatiotemporal Propagation of Epileptiform Activity
  7. Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue
  8. Conclusions
  9. Acknowledgment
  10. References

Epilepsy is a chronic disorder characterized by abnormal spatiotemporal neural activities. To clarify its physiological mechanisms and associated morphological features, we investigated neuronal activities using the flavoprotein fluorescence imaging technique and histopathological changes in epileptogenic tissue resected from patients with epilepsy. We applied an imaging technique suitable for examining human brain slices, and as a consequence achieved sufficient responses with high reproducibility. Moreover, we detected significant alterations in neuronal morphology associated with the acquired responses. Therefore, this strategy is useful for gaining a better understanding of the pathomechanisms underlying intractable epilepsy.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Flavoprotein Fluorescence Imaging in Vitro
  5. Flavoprotein Fluorescence Imaging of Human Brain Slices
  6. Analysis of Spatiotemporal Propagation of Epileptiform Activity
  7. Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue
  8. Conclusions
  9. Acknowledgment
  10. References

Epilepsy is a chronic disorder characterized by abnormal spatiotemporal neural activities. Neurosurgical treatments have been widely applied to patients with drug-resistant intractable epilepsy. Most of the resected specimens containing the epileptogenic focus demonstrate various histopathological features that seem to reflect the abnormal neural activities. Howver, in some instances there is apparent discrepancy between histopathological features and epileptogenic activity. For example, epileptogenicity in focal cortical dysplasia appears to be driven in a different manner from that in cortical tubers of tuberous sclerosis, that is, the former may originate within the lesion in situ,[1] whereas the latter does not originate within the tubers but rather in the peri-tuberous tissue,[2, 3] even though both cortical lesions share characteristic histopathological features. Therefore, to clarify the physiological aspects of the various pathological conditions associated with epilepsy, it would seem informative to investigate the neuronal activities directly using surgical specimens taken from affected patients.

By focusing on tissue resected from humans, several investigators have tried to clarify any characteristic physiological features that are retained in vitro, especially the cells that are responsible for epileptogenesis. For example, in vitro electrophysiological studies of lesions resulting from developmental malformations, including focal cortical dysplasia, using whole-cell patch clamp recordings, have provided information about the unique physiological properties of some morphologically characteristic cells, including dysmorphic neurons and balloon cells.[4] It seems likely that abnormal spreading of neuronal excitation in epileptic patients reflects alterations of neuronal circuitry within the epileptogenic focus. Optical imaging of slice preparations is one of the most appropriate methods for detailed analysis of local neuronal networks because it allows visualization of spatial and temporal relationships over functionally connected areas.

Therefore, to investigate the spatiotemporal dynamics of epileptiform activity, in the present study we performed flavoprotein fluorescence imaging of human brain slices thought to contain the endogenous neuronal circuits responsible for such activity.[5, 6] Here we describe our experimental methods in detail (Fig. 1).

figure

Figure 1. Schematic figure of our study system using human brain slices.

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Flavoprotein Fluorescence Imaging in Vitro

  1. Top of page
  2. Abstract
  3. Introduction
  4. Flavoprotein Fluorescence Imaging in Vitro
  5. Flavoprotein Fluorescence Imaging of Human Brain Slices
  6. Analysis of Spatiotemporal Propagation of Epileptiform Activity
  7. Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue
  8. Conclusions
  9. Acknowledgment
  10. References

Flavoprotein fluorescence imaging is one of several optical imaging methods that exploits activity-dependent changes in flavoprotein fluorescence. Mitochondrial flavoproteins are abundantly present in neurons, and their oxidized form emits green fluorescence (λ = 510–550 nm) under blue light (470–490 nm). Because the change in flavoproteins to their oxidized form is dependent on metabolic activity, monitoring of the resulting change in fluorescence has been used as an indicator of local metabolic changes in brain tissue.[7, 8] Previous studies have shown that changes in flavoprotein fluorescence signals are well correlated with the electrical activities of neurons.[7, 9] Because this technique requires no exogenous dyes, it has none of the disadvantages of dye-related techniques for investigations of spatiotemporal activity in brain slices, such as photobleaching, cellular toxicity and unloading of the dye.[10] Accordingly, this approach ensures high stability and reproducibility for long experimental periods (Fig. 2), which are indispensable requirements for optical imaging of whole large slices of human brain.

figure

Figure 2. A durability test of flavoprotein fluorescence imaging in vitro. (A) A fresh mouse cortical slice was incubated in the recording chamber and a stimulating electrode (indicated by a black dot) placed on it. The same electrical stimulus (± 300 μA; duration 200 μs, 10 Hz for 1 s) was applied at 0, 6, 10, 21, 24 h after the start of the experiment. (A) Images of basal flavoprotein fluorescence before stimulation (left column) and a pseudocolor ratio image at 1.2 s after stimulation (right column). (B) Time course of the signals in the region of interest indicated in A at each time point. Note that approximately the same responses were observed until 10 h from the start of the experiment. After 24 h, the response was still clear, although it was attenuated to about half. image, 0 h; image, 6 h; image, 10 h; image, 21 h; image, 24 h.

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Flavoprotein Fluorescence Imaging of Human Brain Slices

  1. Top of page
  2. Abstract
  3. Introduction
  4. Flavoprotein Fluorescence Imaging in Vitro
  5. Flavoprotein Fluorescence Imaging of Human Brain Slices
  6. Analysis of Spatiotemporal Propagation of Epileptiform Activity
  7. Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue
  8. Conclusions
  9. Acknowledgment
  10. References

The first step in physiological studies using human brain slices is to harvest and transport the tissue while keeping it in good condition (Fig. 1 left). After recording the ECoG (electrocorticogram) as needed, the surgically resected brain tissue is immediately cut into 5-mm pieces in the operating room. Then, tissue samples suitable for physiological experiments or pathological examination are selected, and those for which pathological examination has the highest priority are assigned. Because it is important to use non-damaged tissue as far as possible for physiological experiments, a piece originally positioned centrally in the resected tissue is preferable, rather than one from near the edge. The harvested tissues are immediately immersed in ice-cold artificial cerebrospinal fluid (ACSF) and bubbled with 95% O2 and 5% CO2. The composition of the ACSF (in mmol/L) we employ is: NaCl, 124; KCl, 5; NaH2PO4, 1.24; MgSO4, 1.3; CaCl2, 2.4; NaHCO3, 26; and glucose, 10. The tissues are transported to our laboratory under these conditions within 45 min after removal.

The second step is preparing the brain slices for physiological experiments (Fig. 1 middle). Brain slices 500 μm thick are obtained from the transported brain tissue using a microslicer in our laboratory. Several fresh slices, usually 2–3, are prepared from each brain block. For histological evaluation, residual tissue from the brain block is embedded in optimal cutting temperature compound, and then slices 7 μm thick are prepared using a cryostat (Fig. 3). The sections are stained quickly with HE. Histological features are then compared with the translucent image of the fresh slices. The prepared slices are incubated in ACSF at 29–30°C for more than 1 h to allow recovery from any damage due to the slicing procedure.

figure

Figure 3. Schematic figure showing the use of resected brain tissue.

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The third step is evaluation of the neural activity of the slices. After incubation, each slice is transferred to a submerged recording chamber and perfused continuously with oxygenated ACSF at a flow rate of 1 mL/min. Translucent images taken in infrared light (λ = 930 ± 10 nm) are obtained with a cooled charge-coupled device camera system attached to an inverted epifluorescence microscope to identify the histological architecture. By comparing the microscopic features on the HE sections obtained at the previous step with the translucent image of the fresh slice, the area in which to place the stimulating electrode is determined. This procedure is especially effective for examining neocortical lesions, including focal cortical dysplasia, because otherwise correct orientation of the fresh slices would be difficult to achieve in such cases. The slice is then stimulated electrically and the spatiotemporal activity evaluated in terms of flavoprotein fluorescence imaging every 100–300 ms. Details of the theoretical background of flavoprotein fluorescence imaging have been described previously.[11] Under the experimental conditions employed, responses represented by changes in signal intensity of about 0.5–3% are usually observed. The images obtained are usually averaged eight times to improve their quality; however, a response can be observed even in a single trial (Fig. 4).

figure

Figure 4. Non-averaged flavoprotein fluorescence responses in eight single trials involving repetitive stimulation at 10 Hz for 1 s are overlaid. A clear response can be observed even in single trials, although they show slight unevenness.

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The fourth step is morphological and molecular biological examination to validate the physiological findings (Fig. 1 right). For this purpose, we use a block of brain tissue corresponding to the mirror surface of each of the slices employed for the physiological examination (Fig. 3). These blocks are fixed with 4% paraformaldehyde and embedded in paraffin. This approach allows us to observe microscopic alterations within the blocks. On the other hand, the fresh slice used for optical imaging can also be used for molecular biological study,[6] since the flavoprotein fluorescence method requires no exogenous dye or fixative. Moreover, it is possible to use tissue samples exactly corresponding to the areas exhibiting an abnormal response during flavoprotein fluorescence imaging.

Finally, we integrate all of these findings to gain an overall picture of the mechanism of epileptogenicity.

Analysis of Spatiotemporal Propagation of Epileptiform Activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Flavoprotein Fluorescence Imaging in Vitro
  5. Flavoprotein Fluorescence Imaging of Human Brain Slices
  6. Analysis of Spatiotemporal Propagation of Epileptiform Activity
  7. Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue
  8. Conclusions
  9. Acknowledgment
  10. References

Acquisition of temporally sequential images facilitates three-dimensional analysis of neuronal activity propagation. Previously, we have investigated neocortical tissues that were considered clinically to be the secondary epileptogenic focus, and reported unique propagation of neural activity within the cortical slices.[5] We found that the elicited neural activities spread horizontally along the layers momentarily in the epileptogenic cortex, although they were not observed in control brain tissues taken from patients with brain tumors who had no history of epileptic episodes before surgery (Fig. 5). The characteristic propagation comprises two spatially and temporally unique components: the identically shaped early phase and the polysynaptic late phase. Furthermore, we observed neuronal hypertrophy, loss of dendritic spines, and nodular varicosities of dendrites, which might participate in the aberrant activities observed by flavoprotein fluorescence imaging.

figure

Figure 5. Epileptiform propagation observed in the epilptogenic focus of the cortex. (A) Sequential images of the flavoprotein fluorescence response in non-epileptogenic and epileptogenic cortex elicited by electrical stimulation at layer IV. Time after stimulation is denoted in milliseconds in the bottom right-hand corner. Scale bars: 1 mm. (B) Temporal profiles of the flavoprotein fluorescence signals. A distinguishable early phase (peaks at 320 ms) was observed in the epileptogenic tissue, although this was not clear in the non-epileptogenic tissue. The early phase showed similar responses independent of stimulus intensity, whereas the late phase (peaks at 1120 ms in epileptogenic tissue) and responses of non-epileptogenic tissue paralleled the changes in stimulus intensity.

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Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue

  1. Top of page
  2. Abstract
  3. Introduction
  4. Flavoprotein Fluorescence Imaging in Vitro
  5. Flavoprotein Fluorescence Imaging of Human Brain Slices
  6. Analysis of Spatiotemporal Propagation of Epileptiform Activity
  7. Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue
  8. Conclusions
  9. Acknowledgment
  10. References

Optical imaging is a powerful approach for investigating local neuronal networks in the epileptogenic focus. Previous animal studies using optical imaging in vitro have revealed the topological relationship between the stimulated area and functionally connected area, whereas both areas are topologically apart, such as the thalamus and primary somatosenseory cortex.[12, 13] By applying this type of analysis to human brain slices, we have observed functional connections between heterotopic nodules and the overlying hippocampus.[6] Slices were prepared from the temporal lobe of a 22-year-old man with periventricular nodular heterotopia, who manifested intractable mesial temporal lobe epilepsy. Microscopically, multiple heterotopic nodules were observed adjacent to the subiculum of the hippocampus. We electrically stimulated the incubated slices, and the elicited neural activity was analyzed as changes in flavoprotein fluorescence signals. When we stimulated either the heterotopic nodule or the overlying hippocampus, clear functional coupling of neural activity between these structures was observed (Fig. 6). Interestingly, the functional coupling activities evoked in either the heterotopic nodules or the subiculum showed marked differences in terms of the pharmacological effects of bicuculline. Moreover, using Western blotting, we detected the expression of both NR1 and NR2 (NMDA receptor subunits) in the heterotopic nodules, although at a lower level than in the subiculum. Thus, it seems likely that the excitatory connections between heterotopic nodules and the subiculum involve different mechanisms.

figure

Figure 6. Functional coupling between a heterotopic nodule (HN) and the overlying subiculum (Sb). (A) (a) Macroscopic appearance of the resected tissue, (b) translucent image of the fresh slice, and (c,d,e) flavoprotein fluorescence responses elicited by the HN (c) or Sb (d), after application of bicuculline. (e) A response elicited by the Sb, after application of both bicuculline and 2-amino-5-phosphono-pentanoic acid (APV), an N-methyl –D-aspartic acid (NMDA) receptor antagonist. Note the clear coupling response in HN during application of bicuculline, and its abrogation by APV. (B) Western blot analysis of NR1 and NR2B (both NMDA receptor subunits) expression.

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Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Flavoprotein Fluorescence Imaging in Vitro
  5. Flavoprotein Fluorescence Imaging of Human Brain Slices
  6. Analysis of Spatiotemporal Propagation of Epileptiform Activity
  7. Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue
  8. Conclusions
  9. Acknowledgment
  10. References

Application of the flavoprotein fluorescence imaging technique to human brain slices is useful for investigating the pathomechanisms underlying epileptogenicity. Systematic investigation using morphological and biochemical analysis, as described here, is able to provide useful information on these pathomechanisms. Further investigations utilizing the present methodology may help to clarify the mechanisms underlying other epileptogenic syndromes, including mesial temporal lobe epilepsy, focal cortical dysplasia, and cortical tubers of tuberous sclerosis.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Flavoprotein Fluorescence Imaging in Vitro
  5. Flavoprotein Fluorescence Imaging of Human Brain Slices
  6. Analysis of Spatiotemporal Propagation of Epileptiform Activity
  7. Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue
  8. Conclusions
  9. Acknowledgment
  10. References

This work was supported by Grants-in-Aid (21300134, 22700376) for Scientific Research from MEXT, Japan, a Grant (24-7) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare, Japan, and a Project Research Promotion Grant from the University of Niigata.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Flavoprotein Fluorescence Imaging in Vitro
  5. Flavoprotein Fluorescence Imaging of Human Brain Slices
  6. Analysis of Spatiotemporal Propagation of Epileptiform Activity
  7. Analysis of Neuronal Networks in Slices of Epileptogenic Human Brain Tissue
  8. Conclusions
  9. Acknowledgment
  10. References