Mice are critical model organisms for the study of neuroinflammation. But tissue quantity is often a limiting factor. In a paper published in Methods, researchers describe a protocol for analysis of 33 cytokines and chemokines using only 50 μl of tissue homogenate (Manglani M et al. 2019). The remaining homogenate can be used for RNA extraction or other analysis.
It has been over 60 years since the discovery of interferon alpha, the first known cytokine (Isaacs and Lindenmann 1957). Since then, the discovery and functional elucidation of many other cytokines, chemokines, and growth factors continues to underscore their complex roles in biological systems (Turner et al. 2014), notably immunogenic response to pathogens, intercellular signaling mechanisms, and maintenance of immunological homeostasis. As such, cytokine and chemokine dysregulation continue to be implicated in a number of diseases, including neuroinflammation (Ramesh et al. 2013).
Neuroinflammation can be induced by a number of external factors, such as infection, traumatic brain injury, toxic metabolites, pathogens, microbes, viruses, spinal cord injury, air pollution, and passive smoke. Traumatic brain injury has received increased media attention recently due to its association with both professional and amateur sports and implication in long-term neurological damage. Neuroinflammation can also be induced by host-related factors, including systemic inflammation, autoimmunity, and aging. Neuroinflammatory mechanisms have been implicated in Alzheimer’s disease (Hensley K 2010).
By far the greatest use case for evaluating cytokines and chemokines via multiplex immunoassay panels, such as the Bio-Plex Cytokine and Chemokine Kits, is measuring the circulating cytokines and chemokines via serum or plasma preparation and analysis. In contrast, in order to directly evaluate the local inflammatory markers, the author of a recent article describes a protocol for preparing mouse brain tissue for cytokine and chemokine measurements via the Bio-Plex Mouse Chemokine Panel 33-Plex Assay (Manglani M et al. 2019).
Monitoring cytokine and chemokine concentrations in neurological tissue affords:
- Measuring direct, localized inflammatory markers
- Observing paracrine and juxtracrine cell signaling mechanisms
- Observing the extent of inflammation in relation to the site of trauma (for example, with traumatic brain injury)
In the case of neuroinflammation, local and systemic cytokine and chemokine concentrations differ, as evidenced by different observed concentrations in cerebrospinal fluid compared to plasma and serum (Manglani et al. 2019). In general, correlations between cytokine and chemokine levels in serum in plasma compared to cerebral spinal fluid are not consistently observed (John et al. 2008).
In the 2019 Methods paper, authors describe a protocol for preparing mouse brain tissues to analyze 33 cytokines and chemokines using the Bio-Plex Mouse Chemokine Panel 33-Plex Assay on three cohorts of mice (Plasmodium berghei-infected, Plasmodium berghei-infected/CD8 T-cell–depleted, and naïve control) (Manglani M et al. 2019). P. berghei is a protozoan parasite which, in certain rodents, causes malaria. In a laboratory setting, this parasite can be used to induce cerebral malaria in order to study the numerous damaging effects of neuroinflammation.
The authors demonstrated that mice without T-cell depletion had upregulation of 20 proinflammatory cytokines and chemokines, compared to the naïve controls. In addition, these cytokines and chemokines were significantly reduced in T-cell–depleted mice.
Within the article is an explicit protocol which provides step-by-step instructions for soft tissue sample preparation and measuring the cytokine and chemokine levels using the Bio-Plex Mouse Chemokine Panel 33-Plex Assay. The sample preparation method can be used with other Bio-Plex panels as well.
The workflow described includes:
- Mechanical homogenization using bead-based mechanical homogenizers
- Solubilization and homogenization with the Bio-Rad Bio-Plex Lysis Buffer
- Utilizing lysis buffer co-factors, which include important protease inhibitors
- Running the Bio-Plex Mouse Chemokine Panel 33-Plex Assay
Notably, this sample preparation described in the method papers is also compatible with parallel mRNA analysis via standard qPCR RNA purification schemes (data not shown). The authors suggest that this same methodology can be used for many different tissue types.
Hensley K (2010). Neuroinflammation in Alzheimer’s disease: mechanisms, pathologic consequences, and potential for therapeutic manipulation. J Alzheimers Dis 21, 1–14.
Houser B (2012). Bio-Rad’s Bio-Plex suspension array system, xMAP technology overview. Arch Physiol Biochem 118 (4); 192–196.
Isaacs A and Lindenmann J (1957). Virus interference. I. The interferon. Proc R Soc Lond B Biol Sci 147, 258–267.
John CC et al. (2008). Cerebrospinal fluid cytokine levels and cognitive impairment in cerebral malaria. Am J Trop Med Hyg 78: 198–205.
Manglani M et al. (2019). Method to quantify cytokines and chemokines in mouse brain tissue using Bio-Plex multiplex immunoassays. Methods 158, 22–26.
Ramesh et al. (2013). Cytokines and chemokines at the crossroads of neuroinflammation, neurodegeneration, and neuropathic pain. Mediators Inflamm 2013, 480739.
Turner MD et al. (2014). Cytokines and chemokines: At the crossroads of cell signaling and inflammatory disease. Biochim Biophys Acta 1843, 2,563–2,582.