Neuroinflammation and nociception in a dish

Researchers from the Department of Veterinary Sciences, University of Turin have developed acute and organotypically cultured spinal cord slice preparations (SCSPs) to study pain. This ex vivo Solution enables multiple slices to be obtained from each animal, therefore decreasing the number of animals required per study. The researchers are seeking partners to help further develop and validate the innovative SCSPs for studying nociception and neuroinflammation ex vivo, and for preclinical development of novel therapeutics.

With CRACK IT Solutions funding, Professor Merighi is now working with a team led by Professor Eustace Johnson at the University of Chester, UK. Together, they will use the spinal cord slice platforms model to study the effects of transplanting mesenchymal stem/stromal cells into the spinal cord, to better understand their effects on neuroinflammation, central neuropathic pain and neuronal hyperexcitability.

Controlling pain is a major challenge in clinical medicine. Research in the field of pain and neuroinflammation is currently conducted either in vivo or on isolated cells cultured in vitro. Animal models are used to understand the mechanisms of physiological and/or pathological pain and are always associated with severe suffering. These experiments, usually in rodents, not only require inducing pain by different physical or chemical approaches, but also, specifically when neuropathic or chronic pain is studied, induce the generation of nerve injury resulting in long lasting pain hypersensitivity. Although working in vivo offers the obvious advantage of maintaining an intact neuronal circuitry, the difficulties in using animal models to study the circuitry and neurophysiology of nociceptors are widely documented (Mao,2012). These in vivo models are technically very demanding and suffer from a high degree of variability in the nociceptive responses between individuals and sets of experiments. Additionally, drug bioavailability can be difficult to control and certain substances do not pass the blood-brain barrier, thus requiring spinal intrathecal administration, increasing complexity and welfare burden. Many of these shortcomings can be overcome using cultured primary neurons and/or neuronal cell lines, but circuitries are lost by this approach, rendering it unsuitable to the study of cell-to-cell communication and modulation of neural signals.

To bypass some of these difficulties, the researchers have developed acute and organotypically cultured spinal cord slices and demonstrated that they can provide unique insight into the central mechanisms of nociception and neuroinflammation. The transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1) is crucial to the onset of inflammation of peripheral tissues. The researchers have characterized the response of spinal cord slices to challenge with capsaicin, a natural agonist of TRPV1 (Ferrini et al., 2014), and demonstrated its usefulness to model acute inflammation in central neurons (Frias and Merighi, 2016).

References

  • Ferrini F, Russo A, Salio C (2014). Fos and pERK immunoreactivity in spinal cord slices: Comparative analysis of in vitro models for testing putative antinociceptive molecules. Annals of Anatomy - Anatomischer Anzeiger 196(4): 217-23. doi:10.1016/j.aanat.2013.11.005.
  • Frias B, Merighi A (2016). Capsaicin, Nociception and Pain. Molecules 21(6): 797. doi:10.3390/molecules21060797.
  • Mao J (2012). Current challenges in translational pain research. Trends Pharmacol Sci 33(11): 568-73. doi:10.1016/j.tips.2012.08.001.

The researchers have developed acute and organotypically cultured postnatal (P7-P10) and young adult (P30) spinal cord slice preparations to study pain. The age of the donor is crucial (Jackson et al., 2016) as the nociceptive system matures around P21 in rodents. The possibility to cultivate both immature and mature tissues offers unique opportunities to study the plasticity of neural circuits and their responses to inflammatory challenges. The principles of the preparation and use of the spinal cord slices as a platform for studying central pain signals are illustrated in Figure 1.

Slices have evolved as the predominant in vitro preparation used by electrophysiologists, and to a lesser extent, histologists, pharmacologists and biochemists, because they retain the cytoarchitecture of the tissue of origin. As slice-based assay systems allow precise control of extracellular environments, they facilitate research aiming to establish clear correlations between structure and function, as well as plasticity of neuronal interactions under different experimental conditions.

The cultures can be subjected to inflammatory challenge and the response of the synapses between first and second order nociceptive neurons monitored by bulk-load calcium imaging of their somas, patch-clamp electrophysiological recordings, or quantification of early gene (fos/erk) activation (Salio et al., 2014). The release of nociceptive mediators can be monitored by immunochemical procedures (e.g. ELISA or immunocytochemistry) (Salio et al., 2014). Notably, the system can be further manipulated by genetic engineering using a physical transfection method (gene-gun) that, at least in part, avoids the need to generate novel transgenic strains, further reducing the use of animals in these studies. The researchers have developed an ex vivo FRET-based procedure that monitors caspase 3-dependent neurodegeneration in slice preparations, and can be used to test the effects of sustained inflammation on neuronal survival (Alasia et al., 2015; Lossi et al., 2016).

The system is also amenable to pharmacological medium throughput screening as slices can be co-cultured with a biosensor cell line (“sniffer cell”) expressing receptors for an inflammatory factor of interest (Figure 2). This approach is crucial to test the physiological relevance of centrally released mediators of inflammation as it gives cues on their capability to elicit a functionally relevant response in vivo. Neurons derived from human-induced pluripotent stem cells can also be used as biosensor “sniffer” cells to obtain important translational information on the response of human neurons to slice-derived inflammatory mediators (Ghoochani et al., 2016).

References

  • Alasia S, Cocito C, Merighi A, et al. (2015). Real time visualization of caspase-3 activation by fluorescence resonance energy transfer (FRET). In: Neuronal Cell Death. Methods in Molecular Biology (Methods and Protocols), vol 1254. (Ed. Lossi L, Merighi A), New York: Humana Press/Springer Science+Business Media. doi:10.1007/978-1-4939-2152-2_8.
  • Ghoochani A, Yakubov E, Sehm T, et al. (2016). A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brain. Oncotarget 7(2): 1838-53. doi:10.18632/oncotarget.6550.
  • Jackson SJ, Andrews N, Ball D, et al. (2016). Does age matter? The impact of rodent age on study outcomes. Laboratory Animals 51(2): 160-169. doi:10.1177/0023677216653984.
  • Lossi L, Cocito C, Alasia S, et al. (2016). Ex vivo imaging of active caspase 3 by a FRET-based molecular probe demonstrates the cellular dynamics and localization of the protease in cerebellar granule cells and its regulation by the apoptosis-inhibiting protein survivin. Molecular Neurodegeneration 11(1): 1-20. doi:10.1186/s13024-016-0101-8.
  • Salio C, Ferrini F, Muthuraju S, et al. (2014). Presynaptic modulation of spinal nociceptive transmission by glial cell line-derived neurotrophic factor (GDNF). J Neurosci 34(41): 13819-33. doi:10.1523/JNEUROSCI.0808-14.2014.

Collaborations are sought with academics and/or pharmaceutical and biotechnology companies to:

  • Expand (e.g. by the use of microfluidic culture systems) and validate SCSPs for preclinical drug development and precision medicine.
  • Provide a larger panel of candidate pain-controlling drugs/molecules that could be tested and shortlisted for further development using SCSPs.
  • Explore the utility of the slice model for investigating disease models, for example using the spinal cord of an EAE mouse to study Multiple Sclerosis.

In the framework of a collaborative project/venture, the researchers offer:

  • The possibility to test any compound of interest with the researcher’s SCSPs.
  • The development of slice platforms from other areas of CNS (postnatal cerebellar slices are already routinely in use in the lab) for the study of neurodegeneration.
  • The development of slice platforms from aged brains. 

Using SCSPs has the potential to reduce the number of animals used in experiments by enabling multiple slices to be obtained from each animal, therefore decreasing the number of animals required per study. As an example, 20 slices (400 µm thick) can be easily obtained after dissection of the spinal cord from a P4 mouse. Multiple slices can be cultured in each well of a multi-well plate, enabling different experimental/pharmacological challenges to be performed on SCSPs obtained from the same animals (Figure 3). In an in vivo approach where eight mice were used per group, and four groups studied, SCSPs have the potential to reduce the number of mice used from 32 to 8. The technology also provides a novel medium throughput screening platform amenable to preclinical development of pain-targeted therapeutics.

The number of animals required can be further reduced by avoiding the need to specifically generate new transgenic mouse strains. SCSPs can be transiently transfected to express one or more proteins of interest, removing the requirement to breed transgenic animals for some experiments.

For more information: www.morfovet.it/Merighi/Index.html.

Overview | Publication

Overview

With CRACK IT Solutions funding, Professor Merighi is now working with a team led by Professor Eustace Johnson at the University of Chester, UK. Together, they will use the spinal cord slice platforms model to study the effects of transplanting mesenchymal stem/stromal cells into the spinal cord, to better understand their effects on neuroinflammation, central neuropathic pain and neuronal hyperexcitability. This system has the potential to reduce the number of animals used for these studies by up to 50%, as multiple slices can be cultured from a single animal.

Publication

Lossi L, Merighi A (2018). The Use of ex Vivo Rodent Platforms in Neuroscience Translational Research With Attention to the 3Rs Philosophy. Front. Vet. Sci. 5:164. doi.org/10.3389/fvets.2018.00164.