The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.
Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.
Epilepsy is one of the most common neurological disorders, affecting 1% of the world population, and is associated with increased morbidity and mortality1,2. Unfortunately, one-third of patients suffering from epilepsy are drug-resistant, despite an abundance of available treatment options including more than 20 approved antiepileptic drugs (AEDs)3. Failure to translate results from preclinical animal research to clinical trials is one reason why promising treatment strategies are not effective in many patients4. Recently, neuropeptide Y (NPY) and galanin have been shown to have antiepileptic effects in animal models; though, when tested in resected human brain tissue, only NPY was effective5.
Most of the existing knowledge concerning basic neurological mechanisms and disease therapy approaches stem from animal models and cell culture experiments. Although informative, these models only represent single aspects of complex human diseases and the adult human brain network. Alternatively, human brain tissue has the potential to bridge the translational gap but is rarely available for functional studies. For instance, post mortem brain tissue has been a valuable tool in investigating protein expression, brain morphology, or anatomical connections, though neuronal activity is often compromised is this tissue6,7,8,9,10,11.
In contrast, living resected human brain tissue has been investigated concerning preclinical drug evaluation, basic neuronal functions and gene expression patterns12,13,14,15,16,17. A great advantage of human brain slices compared to rodent slices is the long viability of neuronal tissue after resection and preparation. Compared to rodent brain slices, which can typically be recorded for up to 8 h after preparation, human brain slices show stable neuronal activity for up to 72 h, enabling thorough investigation of these rare and valuable samples12,18.
Several studies have investigated properties of epileptiform activity in various areas of resected cortical and hippocampal human tissue and used different methods for induction of epileptiform activity. In rodent slices, epileptiform activity can be induced by several methods: electrical stimulation of DG hilar cells, increase of extracellular K+ (8–12 mM KCl), blocking of GABAA receptors by bicuculline (BIC), blocking of potassium channels by 4-aminopyridine (4-AP), and removing or reducing Mg2+ in extracellular solution19. However, induction of epileptiform activity in human tissue requires the combination of at least two of the abovementioned methods20,21,22.
Presented here is a method for the preparation of human hippocampal brain slices, which are viable for up to 20 h and show induction of epileptiform activity upon application of high K+ (8 mM) and 4-AP or low Mg2+ and BIC.
Patients must give informed written consent prior to operation, and necessary ethical agreements must be in place prior to the experiment. Concerning the representative results, all studies involving human participants were reviewed and approved by Charité-Universitätsmedizin, Berlin (EA2/111/14).
1. Preparation of 10x solutions
NOTE: Due to difficulties in planning access to human brain tissue, it is recommended to prepare 10x solutions as described here. Alternatively, final 1x solutions can be prepared freshly by adding individual substances in final concentration to double-distilled water (ddH2O).
2. Preparation of 1x final solutions
NOTE: Final 1x solutions should be prepared fresh or earliest as possible on the day before use. All final solutions should be carbogenated with 5% CO2 and 95% O2 using a glass gas disperser to enrich solutions with oxygen, and adjust the pH to 7.4 (max = 7.4 ± 0.2).
3. Preparation of interface chamber
4. Set-up of preparation area
NOTE: Preparation can be performed under sterile conditions to avoid contamination and elongate slice survival. However, not all vibratomes fit under a sterile hood, and other measures are required to reduce contamination during preparation. This section describes some of these measures.
5. Tissue slicing and storing
6. Recording of epileptiform activity
7. Analysis
Epileptiform activity has been successfully recorded in resected human hippocampal tissue originating from up to 15 patients. Establishing stable transport and preparation procedures is critical for successful induction of epileptiform activity in human brain tissue. Recently published results have shown 1) stable induction of epileptiform activity in resected tissue of different patients as well as 2) the use of resected human brain tissue as a preclinical tool for evaluation of novel antiepileptic mechanisms14,20.
Application of highK++4-AP induced epileptiform activity in form of burst activity within a few minutes (Figure 2A,B,C,D). Due to low neuronal distribution in human hippocampal tissue or high neuronal cell loss due to temporal lobe epilepsy (TLE), placement of electrodes can be adjusted in the beginning of recording. In cases where burst activity of slices is not visible in the CA1 area after 10 min (independent of electrode placement), slice viability may be compromised, and the slice will need to be replaced.
SLEs, with a duration of >10 s, can be induced with application of lowMg2++BIC (Figure 2E,F). Figure 2E shows stable induction of SLEs after a few minutes and stable frequency throughout the recording. Here, SLE activity was successfully induced in two of four slices from the investigated patient. One slice showed only burst activity after 15 min of SLE activity, whereas the other slice did not show SLEs even after 40 min.
For preclinical evaluation of substance effects, a potential antiepileptic effect on burst activity induced by highK++4-AP was investigated. Known and potential antiepileptic substances (lacosamide, DMEA, dynorphine14) were tested, and examples are shown here for the conventional AED lacosamide (a sodium channel blocker) as well as DMEA (a novel potential antiepileptic substance)20. The number of events and inter-event interval (IEI) of burst events decreased both during application of lacosamide and DMEA (Figure 3C), though amplitudes were mostly unaffected (Figure 3D). In a subset of slices, even though induction of burst events was achieved in the first few minutes, the frequency of activity did not recover during wash out of applied AEDs (data not shown here, see Kraus et al.20). Here, the applied drugs were considered to induce effects; however, decreases in burst activity may have been affected by the gradual decay in activity during long recordings. Thus, results must be interpreted carefully.
Figure 1: Interface chamber. For storing of human hippocampal brain slices, an interface chamber with two brain slice holding compartments is used (A); specifically, a Haas-type interface chamber23. Here, hippocampal brain slices rest on (d) three layers of filter paper, (e) smaller pieces to enable handling of individual brain slices, and (f) bigger filter paper pieces to ensure a sufficient layer of solution below the slice. (c) A cotton string surrounding the brain slices, on top of the filter papers, ensures even solution flow from the inlets at the (a) top of the compartment. (b) A cover lid directs oxygen from below the compartment onto the slice. (B) Top view of one slice-holding compartment. (C) Side view to illustrate the layers of filter papers. (g) Bottom of the chamber. (h) Tube for solution inflow, which is connected to a peristaltic pump (blue arrows mark the direction of the solution flow). Please click here to view a larger version of this figure.
Figure 2: Epileptiform activity in human hippocampal slices induced by highK++4-AP and lowMg2++BIC. CA1 example recordings and excerpts of application of highK+ (8 mM)+4-AP (100 µM) (A,B,C,D) and lowMg2++BIC (10 µM) (E,F). (A) Bath application of highK++4-AP induces epileptiform activity within a few minutes, and activity is stable for at least 60 min. Details of (A) can be seen in (B). Two different types of activity are induced in the CA1 area of human hippocampal slices: interictal-like spikes (C, details of [B]) and burst activity (D, details of [B]). Burst activity was shown to be sensitive to antiepileptic drugs and therefore analyzed for the effect of potential antiepileptic substances (Figure 3). (E,F) Application of lowMg2++BIC induces SLEs at a duration of >10 s (F) in CA1 within a few minutes. However, induction of SLEs can take up to 30 min in other slices. Scale bars = 0.2 mV, 2 min (A,E), 5 s (B), 500 ms (C,D), 5 min (E), and 2 s (F). This figure has been adapted from Kraus et al.20. Please click here to view a larger version of this figure.
Figure 3: Decrease in epileptic burst activity of human slices during application of lacosamide or DMEA. Burst activity decreased during application of (A) lacosamide and (B) DMEA, a potential new antiepileptic molecule. (A) and (B) show exemplary recordings of the CA1 area with excerpts of regions used for analysis in (C) and (D). Burst activity decreased during lacosamide (100 µM) and DMEA (10 mM) application, as seen by middle excerpts and increases again during wash out. (C,D) Number and amplitude of burst activity were analyzed for the last 5 min of each application phase (baseline, lacosamide/DMEA, wash out) and shown as summarized results for all patients (number of events, C; amplitude, D) as mean ± SD. Each dot indicates one patient. Asterisks mark significant differences as assessed by either Friedman test and post-hoc with Dunnett's multiple comparison of groups for analysis of lacosamide application (*p < 0.05, n = 4) or by repeated measurement ANOVA and post-hoc with Tukey’s comparison for analysis of DMEA application (**p < 0.01, n = 10). Scale bars = 0.2 mV, 2 min (full recording, A), 5 s (excerpts, A), 3 min (full recording, B), and 1 s (excerpts, B). This figure has been adapted from Kraus et al.20. Please click here to view a larger version of this figure.
Solution 1.1 choline aCSF | |||
Substance | 10x concentration (mM) | 1x concentration (mM) | Note |
choline Cl | 1100 | 110 | |
(+)-Na L-ascorbate | 116 | 11.6 | |
MgCl2x6H2O | 70 | 7 | |
Na pyruvate | 31 | 3.1 | |
KCl | 25 | 2.5 | |
NaH2PO4 | 12.5 | 1.25 | |
NaHCO3 | 260 | 26 | |
CaCl2 | – | 0.5 | add to final solution |
Glucose | – | 10 | add to final solution |
Solution 1.2 aCSF | |||
Substance | 10x concentration (mM) | 1x concentration (mM) | Note |
NaCl | 1290 | 129 | |
NaH2PO4 | 12.5 | 1.25 | |
CaCl2 | 16 | 1.6 | |
KCl | 30 | 3 | |
MgSO4 | 18 | 1.8 | |
Glucose | – | 10 | add to final solution |
Solution 1.3 highK++4-AP aCSF | |||
Substance | 10x concentration (mM) | 1x concentration (mM) | Note |
NaCl | 1240 | 124 | |
NaH2PO4 | 12.5 | 1.25 | |
CaCl2 | 16 | 1.6 | |
KCl | 80 | 8 | |
MgSO4 | 18 | 1.8 | |
Glucose | – | 10 | add to final solution |
4-AP | – | 0.1 | add to final solution |
Solution 1.4 lowMg2++BIC aCSF | |||
Substance | 10x concentration (mM) | 1x concentration (mM) | Note |
NaCl | 1300 | 130 | |
NaH2PO4 | 12.5 | 1.25 | |
CaCl2 | 16 | 1.6 | |
KCl | 30 | 3 | |
Glucose | – | 10 | add to final solution |
BIC | – | 0.01 | add to final solution |
Solution 2 | |||
Substance | 10x concentration (mM) | 1x concentration (mM) | Note |
NaHCO3 | 210 | 21 |
Table 1: Preparation of 10x and final 1x solutions for transport, preparation, and recording.
Living resected human brain tissue is a highly valuable tool in preclinical evaluation of AEDs, as it properly represents an intact human brain micro-network. The presented protocol describes a method for tissue transport and preparation, which ensures high quality hippocampal slices as well as a stable induction method for epileptiform activity critical for AED evaluation.
Investigation of epileptiform activity as well as methods for chemical or electrical induction in human brain slices have been previously shown by other groups17,20,21,22. This protocol describes the induction of stable burst activity in slices from different patients via application of high K++4-AP as well as induction of SLEs in CA1 area via application of low Mg2++BIC. It was found that the induction of burst activity is more consistent (80% of tested slices in 15 patients) than the induction of SLEs (50% of tested slices in one patient). However, thus far, the induction of SLEs has only been testd in one patient. Nevertheless, induction of SLEs by low Mg2++BIC is recommended, as SLEs have not yet been able to be induced using high K++4-AP.
Several studies have introduced methods for transport and preparation of human brain tissue and often highlight three factors critical to neuronal survival: transportation time, used transport solutions, and storing conditions.
For optimal slice viability, some groups suggest that the transport of resected brain tissue be as short as possible. However, operation rooms and laboratories are rarely in close proximity, meaning that slice quality may be compromised due to long transportation. Some groups have overcome this obstacle by applying constant O2 to the solution during transport12. We have transported brain tissue for short (max = 15 min) and long (up to 1 h) periods of time without constant additional O2 supply during transport, similar to other groups18,25. In these cases, differences in tissue quality were not observed during epileptiform recordings. In communication with other groups at our institute, slice quality did not change for patch-clamp experiments, either. In contrast, variance in tissue quality possibly stems from damage during operations, prolonged resection, and slicing procedure.
Concerning transport and cutting solution, all published methods omit NaCl from solutions to reduce cell swelling due to osmotic pressure, similar to the standard procedure for rodent patch-clamp experiments. However, several substitutes have been introduced so far (i.e., sucrose based aCSF13,22, NMDG-based aCSF12,26, and choline-based aCSF27). Ting and colleagues introduced the NMDG-based aCSF for slice preparation in 201426 and later added a recovery protocol, which slowly reintroduces NaCl to the slices28. However, as described by Ting et al., neurons of brain tissue prepared in NMDG-based aCSF show higher membrane resistance, thus affecting whole-cell seal during patch-clamp experiments26. Therefore, we have transitioned from NMDG-based aCSF to the use of choline-based aCSF20, which yields high quality slices for both field potential and patch-clamp recordings.
Concerning storage of slices, it is generally accepted that interface conditions provide optimal oxygenation critical for long slice survival18. However, other groups show slice survival for up to 72 h under submerged conditions12. Contrary to previous hypothesis, human brain slices seem to be more resistant to low oxygenation or oxidative stress compared to rodent slices. Primarily, interface chambers have been previously used for storing of human hippocampal slices, though submerged conditions are recommended for the maintenance of human brain slices in patch-clamp experiments.
As discussed by other groups, an additional critical step for long slice survival (interface for <48 h18, submerged for <72 h12) is the prevention of bacterial contamination. Rodent brain slices are typically used in electrophysiological recordings for up to 8 h, and bacterial contamination is not considered to affect slice viability during this period. High number of slices prepared from one resection and the uncommon availability of human brain tissue highlights the need to prolong viability of human brain slices. This method successfully describes the preparation of living human hippocampal brain slices, which can easily be adapted to sterile conditions. However, for the recordings performed here, slice survival extending 20 h was not a priority.
Recording in interface chambers has also been shown to be essential for induction of epileptiform activity such as SLEs22. Submerged conditions, due to low oxygenation, are rarely used for recording of SLEs; though, they are necessary for optical high resolution needed for patch-clamp experiments. The use of an optimized submerged type recording chamber enables the recording of epileptiform activity (extracellular field or single neuron) in human brain slices, due to high oxygenation and fast drug application29. Here, methods and results for field potential recordings are described, but it should be emphasized that patch-clamp recordings have been successfully performed in mouse and human brain slices using this modified recording chamber (data not shown).
Resected human brain tissue has a higher translational value compared to rodent models. It represents an adult, diseased neuronal network that cannot be reproduced by iPSCs. However, as in any in vitro system, human brain slices do not represent an intact human brain. Additionally, the recorded neuronal networks of resected brain tissue can undergo substantial molecular and functional changes due to damage during operation or preparation. Slicing procedures have been shown to affect GABAergic function and may affect the induction of epileptiform activity30. These limitations should be considered while formulating a hypothesis. When testing potential antiepileptic drugs, the use of different brain areas should be considered, as drug targets might not be expressed in all human brain regions or all patients. In particular, the hippocampi of TLE patients often show signs of hippocampal sclerosis accompanied by severe neuronal cell loss. It is recommended to obtain patient information on pathological changes and disease history, such as potential refractory towards medications, and consider this during data interpretation.
In conclusion, this method successfully describes the preparation of living human hippocampal brain slices and induction techniques for recording two different types of epileptiform activity. Since the availability of living human brain tissue is rare, optimized transport and recording conditions should be used to ensure maximum output from experiments using human brain slices. It is suggested that resected human brain tissue can be used as a preclinical validation tool in addition to rodent models and cell culture experiments.
The authors have nothing to disclose.
We thank Mandy Marbler-Pötter (Charite-Unversitätsmedizin, Berlin) for excellent technical assistance. P.F. was funded by the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft) under Germany´s Excellence Strategy-EXC-2049-390688087. This work has been supported by the QUEST Center for Transforming Biomedical Research at the Berlin Institute of Health.
(+)-Na L-ascorbate | Sigma Aldrich | A4034 | |
4-AP | Sigma Aldrich | 275875-5G | |
Blades | eliteSERVE GmbH | HW3 | used for the vibratome |
CaCl2 | Merck | 102382 | |
Choline Cl | Sigma Aldrich | C1879 | |
Filter paper | Tiffen | EK1546027T | |
Gas-tight bottle caps | Carl Roth GmbH+Co.KG | E694.1 | |
Glass filaments | Science Products | GB150F-8P | for recording electrodes |
Glass gas disperser | DWK Life Sciences GmbH | 258573309 | |
Glucose | Sigma Aldrich | G7528 | |
Interface Chamber | inhouse made | – | see Haas et al., 1979 |
KCl | AppliChem | 131494.1210 | |
Membrane (Cell culture inserts) | Merck | PICM030050 | |
Membrane chamber | inhouse made | – | see Hill and Greenfield, 2011 |
MgCl2∙6H2O | Carl Roth | HNO3.2 | |
MgSO4 | Sigma Aldrich | M7506 | |
Na pyruvate | Sigma Aldrich | P8574 | |
NaCl | Carl Roth | 3957.1 | |
NaH2PO4 | Merck | 106346 | |
NaHCO3 | Carl Roth | HNO1.2 | |
Peristaltic pump | Gilson | Minipuls 3 | |
Slice holder | Warner instruments | SHD-41/15 | |
Vertical puller | Narishige | PC-10 | |
Vibratome | Leica | VT1200S |