Analysis of the immune response to sciatic nerve injury identifies efferocytosis as a key mechanism of nerve debridement

Abstract

Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6Chigh monocytes infiltrate the nerve first, and rapidly give way to Ly6Cnegative inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages 'eat' apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF deficient (Csf2-/-) mice, inflammation resolution is delayed and conditioning-lesion induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion induced neurorepair.

Transcriptional profiling of mouse peripheral nerves to the single-cell level to build a sciatic nerve ATlas (SNAT)

“…We have integrated our data in a freely accessible and searchable web interface ( https://www.snat.ethz.ch ) with the intention to make a contribution to the filling of a gap in current peripheral nerve research. Using this resource as baseline, together with recently published complementary extensions mainly focusing on immune cells in peripheral nerves ( Kalinski et al, 2020 ; Wang et al, 2020 ; Ydens et al, 2020 ) and adult nerves ( Wolbert et al, 2020 ), we anticipate that there will be expanding efforts to cover various aspects of peripheral nerve biology such as in cellular-, injury-, aging-, and disease models ( Toma et al, 2020 ) as well as covering other related glia cells ( Avraham et al, 2020 ; Stierli et al, 2019 ), hopefully yielding insightful and valuable data as the fundamental basis of building and testing hypothesis in functional follow-up studies.…”

Interactions Among Nerve Regeneration, Angiogenesis, and the Immune Response Immediately After Sciatic Nerve Crush Injury in Sprague-Dawley Rats

“…Axonogenesis, angiogenesis and the immune response have numerous interactions after nerve injury ( Cattin et al, 2015 ). In order to better verified cell clusters involved in the nerve injury and repair process, Kalinski et al (2020) performed single-cell RNA sequencing in mice sciatic nerves after crush injury models. After being injured for 3 days, 24 different cell clusters were identified.…”

Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release

“…Prior to cryostat sectioning, first place the DRG in OCT and keep for at least 18 h or longer time periods at −80°C. OCT frozen DRGs are now ready for cryostat cutting and staining ( Hill et al., 2018 ; Kalinski et al., 2020 ). Note: Perfusion of the mouse with 4% paraformaldehyde instead of PBS, prior to dissection, improves the downstream immune fluorescence and can be performed if no DRGs are collected for live cell applications.…”

“…Prior to cryostat sectioning, first place the DRG in OCT and keep for at least 18 h or longer time periods at −80°C. OCT frozen DRGs are now ready for cryostat cutting and staining ( Hill et al., 2018 ; Kalinski et al., 2020 ).…”

“…The protocol describes the dissection of DRGs from the whole accessible spine of a mouse. It does not describe the dissection of the DRG from certain spinal segments that might be needed for studies on peripheral nerves (e.g., sciatic nerve) ( Kalinski et al., 2020 ; Swett et al., 1991 ). Also, there is no description of the dissection of the trigeminal ganglion provided ( Katzenell et al., 2017 ), which contains the cell bodies of the sensory neurons that innervate the head and face.…”