We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.
Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-α to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.
Sterile and aseptic techniques are critical to the preparation, maintenance, and use of slice cultures for extended periods. Furthermore, the rationale for our use of slice cultures only after 18 days in vitro is based on evidence that indicates excitatory and inhibitory synaptic transmission becomes most mature, and glia (astrocytes and microglia) become quiescent and consistent with their in vivo counterparts (Figure 1).
1. Preparation & Maintenance of Hippocampal Slice Cultures
2. Pre-screen Vitality of Slice Cultures
3. Cold-Preconditioning
4. Excitotoxic Injury
5. Co-treatments Applied with Cold-preconditioning
An important advantage of slice cultures is that environmental conditions can be accurately controlled. This means that cytokine signaling from cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects.
6. Immediate and Delayed Excitotoxic Injury after Cold-preconditioning
7. RNA Isolation
The following gene expression procedures are scaled for a single slice culture.
8. RNA Quantification
9. SYBR Green Quantitative PCR
10. Quantitative PCR for Microarrays
Quantitative real-time qPCR array screening is a highly sensitive and reproducible means to probe for low-level inflammatory mediator expression changes.
11. Multiplexed Microsphere Flow Cytometric Proteomic Assay
12. Immunohistochemistry
13. Representative Results
Figure 1. Appearance of hippocampal slice cultures and microglia. The left hand image shows a typical mature (i.e., 21 day in vitro) hippocampal slice culture stain with NeuN (green) to illustrate the cytoarchitecture of the principal neurons. Pyramidal neurons are shown in areas CA1 and CA3 and dentate gyrus (DG) neurons to the left. Scale bar is 250 μm. The right hand image is derived from the CA1 area and shown at higher power to illustrate the branched quality of quiescent microglia within mature slice cultures. Cells were marked with the microglial surface marker, cd11b. Scale bar is 50 μm.
Figure 2. Cold-preconditioning neuroprotection in hippocampal slice cultures. Cultures are incubated with Sytox Green, a fluorescent marked dead cell marker. The top row shows sham control cultures and the bottom row shows slices exposed to 28°C for 90 min. Left hand, pre-screen images show no CA1 injury. Relative injury color calibration scale is shown in the left upper image. Middle row images show relative slice culture injury 24 hours after exposure to 20 μM NMDA. Notice that sham control injury is greater than that of cultures exposed to cold-preconditioning (CP). Traditionally, cultures are then exposed to 20 μM NMDA overnight to maximize CA1 neuronal injury levels and relative injury of CP v. sham, noted as a ratio of injury/maximal injury. However, exposure to maximal injury stimuli may not be sufficient to overcome neuroprotection from preconditioning. Accordingly, use of ratios of injury/ maximal injury may not accurately reflect neuroprotection from preconditioning. This is evident in the right hand images that show CP maximal levels are less than those of the sham controls.
Figure 3. Schematic illustrating the utility of using initial, first day measurements to quantitate injury levels in cold-preconditioning experiments. As noted above, we found that use of ratios (injury/maximal injury) could obscure neuroprotection from cold-pre-conditioning. This can be seen from the schematic to the left where sham injury is shown in red and that after cold-preconditioning in blue. One day after NMDA exposure cold-preconditioning shows a relative “3” level of injury v. sham (“5”) control, consistent with 40% neuroprotection. However, if a traditional format using ratios (i.e., injury/maximal injury) is used, no protection is evident [i.e., (5/10)=50% for sham v. (3/6)=50% for cold-preconditioning].
Figure 4. Typial qPCR result are shown. Upper curve RNA copy number v. threshold cycle for PCR amplification showing controls (blue) and experimental samples (red). Lower image shows typical amplification profiles for four samples (black, green, yellow and purple). Notice the latter Ct threshold cycles (marked by orange line) occur at 26.0, 29.5, 31.0, and 32.5.
Figure 5. Double-label immunostaining used to confirm qPCR and qPCR array results. A slice culture was processed for IL-11 (red) and NeuN (green; to mark neurons) to probe for the cellular expression loci of IL-11. Notice that some pyramidal neurons (arrows) show IL-11 and NeuN staining (yellow) while a few smaller cells (arrows), presumed to be astrocytes, show only increased IL-11 staining (red).
Two fundamentally important concepts important to delineation of the cytokine signaling system involved in cold-preconditioning neuroprotection are illustrated in Figures 6 and 7. First, cytokines are extremely low concentration signaling molecules in normal brain. Nonetheless, physiological cytokine concentration changes have an immense potential to alter brain structure and function (i.e., phenotype) because of their ability to alter gene expression (Figure 6). Furthermore, cytokines are highly redundant and pleiotropic in that multiple cytokines can have similar effects and a single cytokine can have variable effects (Figure 7). Thus, to accurately establish the innate cytokine-bases for neuroprotection from cold-preconditioning (or other physiological preconditioning stimuli), composite analysis of related signaling variables must be determined. This is accomplished via multiplexed assay strategies. This will establish the cytokine “signature” of cold-preconditioning neuroprotection.
Figure 6. Power of brain cytokine signaling. The illustrations convey the immense signaling power of physiological concentrations of brain cytokines compared to the concentrations of other well-recognized counterparts. Concentration is represented as the inverse of distance, beginning with a single cat whisker as the reference point. Sodium (10-1M illustrated by a grain of pepper) and potassium (10-3M illustrated by a tomato) are present in the interstitial brain space at levels of around 150 and 3 mM, respectively, and have well-recognized roles in neural cell electrophysiological function. Similarly, pH (i.e., ~10-7M levels of hydrogen ions and illustrated as 780 meters along Chicago’s lakefront) and calcium (i.e., ~10-8M levels shown as 7.8 km seen from a satellite Google image along Chicago’s lakefront from McCormick Place to Promontory Point in Hyde Park near the University of Chicago). In addition, neurotransmitters released to interstitial space over these concentrations affect local brain region activity. In contrast, cytokines (shown as the distance from Earth to Mars) can alter brain function at concentrations more than ten million times less.
Figure 7. Interactive signaling of innate cytokine pathways. Cytokines are highly redundant and pleiotropic in that multiple cytokines can have similar effects and a single cytokine can have variable effects. Such diversity stems from complex interactive signaling that occurs at the level of ligands, receptors, and phosphoproteins. Further complexity stems from the fact that brain consists of different cell types, with each capable of cell-specific innate cytokine, receptor, and related phosphoprotein changes. For illustrative purposes here, only innate cytokine signaling pathways (derived from immune cell studies) are shown. For simplicity, the brain is drawn as a single cell (white line) showing the potential interactions for innate cytokines (IL-1α and IL-1β (referred to here as IL-1α/β), TNF-α, IL-6, IFN-γ and IL-10), receptors (IL-1R1, TNFR1, IL-6/gp130, IFNγR, and IL-10R), and phosphoproteins (i.e., kinases ERK1/2, P38 (P38-MAPK) and JNK) and transcription factors (ATF-2, NFκB, and STAT3). For example, TNF-α signals via TNFR1 to JNK, p38-MAPK and ERK1/2, which triggers gene expression via ATF-2. TNF-α also alters gene expression directly through NFκB activation. Together, activation of these transcription factors evoke increased (arrow) and decreased (blunt end) expression of cytokines and their receptors as indicated. Importantly, these pathways show that changes in one cytokine (e.g., TNF-α) influence production of other (e.g., IL-1β) cytokines. Thus, to establish accurately the cytokine-bases for neuroprotection from cold-preconditioning, composite analysis of related signaling variables must be determined. This will establish the innate cytokine “signature” of cold-preconditioning. (Image was compiled from data of reference #25.)
The authors have nothing to disclose.
This work was funded by grants from the National Institute of Neurological Disorders and Stroke (NS-19108), the Migraine Research Foundation, and the White Foundation to Richard P. Kraig. Ms. Marcia P. Kraig assisted in preparation of culture media and maintenance of slice cultures. We thank Yelena Grinberg for her comments and revisions on a final version of this article.
Preparation of Slice Cultures
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
Millicell-CM tissue culture inserts (30 mm) | Thermo Fisher | PICM0-3050 | |
Basal Medium Eagle | Invitrogen | 21010 | |
Earle s Balanced Salt Solution | Sigma | E2888 | |
Horse serum | Invitrogen | 26050-088 | |
Glutamax | Invitrogen | 35050 | |
Gentamicin | Invitrogen | 15710-064 | |
Fungizone | Invitrogen | 15295 | |
D-glucose | Sigma | G8769 | |
BSL-1 fume hood | Baker | ||
McIlwain tissue chopper | Brinkmann | ||
Teflon disks | Brinkmann | 023-49-310-1 | |
Spatula | Thermo Fisher | 14-373-25A | |
Curved iris scissors | Fine Science Tools | 14091-09 | |
Long straight scissors | Fine Science Tools | 14002-14 | |
Long forceps | Thermo Fisher | 13-812-40 | |
Angled forceps | Fine Science Tools | 11808-02 | |
Short forceps | Thermo Fisher | 13-812-38 | |
#5 Inox forceps | Fine Science Tools | 11254-20 | |
2 Iris spatulae | Fine Science Tools | 10094-13 | |
150 x 15 mm Petri dishes | Thermo Fisher | 08-757-148 | |
60 x 15 mm Petri dishes | Thermo Fisher | 08-757-100B | |
Wild M8 stereomicroscope | Wild Heerbrugg | ||
Gey s balance salt solution | Sigma | G9779 | |
Blak-Ray shortwave Ultraviolet measuring meter | UVP | J-225 | |
6-well Falcon culture dishes | Thermo Fisher | 08-772-1B | |
Neurobasal | Invitrogen | 21103 | |
B27 supplement | Invitrogen | 17504 | |
Gem21 Neuroplex | Gemini Bioproducts | 400-160-010 | |
Ascorbic acid | Sigma | A4544 | |
CO2 Analyzer | Bacharach | #2820 |
Pre-screening for Vitality of Slice Cultures
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
Sytox Green | Invitrogen | S7020 | |
DMIBRE inverted microscope | Leica |
Cold-Preconditioning
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
BSL-2 Fume Hood | NuAire |
Excitotoxic Injury
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
CoolSnap fx CCD camera | Photometrics | ||
Fluoroscein | Invitrogen | F36915 | |
MetaMorph software | Molecular Devices | v. 7.5.04 | |
100 μm deep hemacytometer | Hausser Scientific | ||
N-methyl-D-aspartate | Calbiochem | 454575 | |
SigmaStat software | Systat Software Inc. | v. 3.5 |
Co-treatments applied with Cold-Preconditioning
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
sTNFR1 | R & D Systems | 425-R1-050 | |
Bovine Serum Albumin | Sigma | A-4503 |
RNA Isolation
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
RNAlater | Ambion | AM7021 | |
Micro RNEasy Kit | Qiagen | 74004 | |
β-mercaptoethanol | Fisher | 03446-100 | |
Diposable 1.5 RNAse-free Pestle |
South Jersey Precision Tool and Mold, Inc. | 749521-1590 | |
RNasin | Promega | N261A | |
Tris[hydroxymethyl]aminomethane | Sigma | T3069 | |
EDTA (ethylenediaminetetraacetic acid disodium salt hydrate | Sigma | #E7889 |
RNA Quantification
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
RiboGreen Assay | Invitrogen | R11491 | |
Yeast tRNA | Invitrogen | 15401-011 | |
1.5 mL centrifuge tubes | USA Scientific | 16155500 | |
96-well fluorescent assay plate | Nunc | DK-4000 | |
96-well fluorescent plate reader | Tecan | Safire II |
Quantitative PCR
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
iScript reverse transcription kit | Bio-Rad | 170-8891 | |
SYBR Green | Invitrogen | S7563 | |
Platinum Taq polymerase | Invitrogen | RNA Ultrasense 11732-927 | |
iCycler thermocycler | Bio-Rad | iCycler Optical Module | |
iCycler system software | Bio-Rad | v. 3.1 |
Quantitative PCR for Microarrays
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
RNase Away | Molecular BioProducts | #7000 | |
RT2 First Strand Kit | SABiosciences | C-03 | |
SYBR Green-based PCR mix | SABiosciences | PA-011 | Specific for iCycler |
RT2 Profiler PCR Array | SABiosciences | PARN-011A |
Proteomic Assay
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
Cell lysis kit | Bio-Rad | 171-304011 | |
Microcentrifuge | Fisher Scientific | Micro 7 | |
BSA protein stock | Bio-Rad | 500-0007 | |
BCA protein assay kit | Thermo Scientific | 23225 | |
Immuno 96 MicroWell plates | Nunc | 449824 | |
Sealing tape | Bio-Rad | 2239444 | |
96-well plate reader | Perkin Elmer | 1420-mulilabel counter | |
Bioplex Rat TNF-α cytokine assay | Bio-Rad | X0000001T |
Immunostaining
Name of Reagent/Equipment | Company | Catalogue/Model Number | Comments |
16% Paraformaldehyde | Alfa Aesar | 43368 | |
Lysine | Sigma | L-6001 | |
Sodium phosphate | Fisher Scientific | S374-1 | |
Sodium periodate | Sigma | S-1878 | |
Sodium azide | Sigma | S-2002 | |
Hydrogen Peroxide (30%) | Sigma | H1009 | |
Goat serum | Colorado Serum Company | CS 0920 | |
Triton X-100 | Sigma | T-8787 | |
Staining dishes | Ted Pella, Inc. | 14511 | |
Plate Shaker | Lab-Line Instruments, Inc. | 3520 | |
#1 Filter Papers (185 mm) | Whatman | 1001-185 | |
3,3 -diaminobenzidine | Sigma | D8001 | |
Dimethyl sulfoxide | Sigma | D8418 | |
Ethanol | Pharmco-AAPER | 111000200 | |
Xylene | Fisher Scientific | X3S-4 | |
Permount mounting media | Fisher Scientific | SP15-100 | |
Cryostat | Leica | cm3050 S | |
Tissue-Tek | Ted Pella, Inc. | 27050 | |
Fine-tip paint brush | Ted Pella | 11806 | |
PAP blocking pen | Ted Pella, Inc. | 22309 | |
SFX signal enhancer | Invitrogen | A31631 | |
ProLong Antifade | Invitrogen | P7481 |