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.

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No conflicts of interest declared.

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.
<img src="/files/ftp_upload/2192/2192fig6.jpg" alt="Figure 6" /> 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.
<img src="/files/ftp_upload/2192/2192fig7.jpg" alt="Figure 7" /> 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-1&alpha; and IL-1&beta; (referred to here as IL-1&alpha;/&beta;), TNF-&alpha;, IL-6, IFN-&gamma; and IL-10), receptors (IL-1R1, TNFR1, IL-6/gp130, IFN&gamma;R, and IL-10R), and phosphoproteins (i.e., kinases ERK1/2, P38 (P38-MAPK) and JNK) and transcription factors (ATF-2, NF&#954;B, and STAT3). For example, TNF-&alpha; signals via TNFR1 to JNK, p38-MAPK and ERK1/2, which triggers gene expression via ATF-2. TNF-&alpha; also alters gene expression directly through NF&#954;B 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-&alpha;) influence production of other (e.g., IL-1&beta;) 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.)

strategies for study of neuroprotection from cold-preconditioning

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-&alpha; 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.

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Strategies for Study of Neuroprotection from Cold-preconditioning

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 ...

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14.7K Views.  The University of Chicago Medical Center. 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. 