Detection of local and remote cellular damage caused by spinal cord and peripheral nerve injury using a heat shock signaling reporter system

Abstract

Spinal cord and peripheral nerve injury results in extensive damage to the locally injured cells as well as distant cells that are functionally connected to them. Both primary and secondary damage can cause a broad range of clinical abnormalities, including neuropathic pain and cognitive and memory dysfunction. However, the mechanisms underlying these abnormalities remain unclear, awaiting new methods to identify affected cells to enable examination of their molecular, cellular and physiological characteristics. Here, we report that both primary and secondary damage to cells in mouse models of spinal cord and peripheral nerve injury can be detected in vivo using a novel fluorescent reporter system based on the immediate stress response via activation of Heat Shock Factor 1. We also provide evidence for altered electrophysiological properties of reporter-positive secondarily-injured neurons. The comprehensive identification of injured, but surviving cells located both close and at distant locations from the injury site in vivo will provide a way to study their pathophysiology and possibly prevention of their further deterioration.

Keywords: Cellular damage; DRG, dorsal root ganglion; FG, Fluoro-Gold; HRP, horseradish peroxidase; HSE, heat shock-response element; HSF1, heat shock factor 1; HSP, heat shock protein; Heat shock signaling; IL-6, interleukin 6; M1, primary motor cortex; M2, secondary motor cortex; MPtA, medial parietal association cortex; PBS, phosphate buffered saline; PCR, polymerase chain reaction; RFP, red fluorescent protein; Reporter mouse; SCI, spinal cord injury; SNI, sciatic nerve injury; Sciatic nerve injury; Spinal cord injury; WDR, wide-dynamic range; WGA, wheat germ agglutinin.

Fig. 1

Design of the HSE-RFP reporter construct. (A) The reporter transgenic mouse harboring the Heat Shock-response Element (HSE) that contains the mouse Hsp70 promoter followed by RFP (DsRed2) coding sequence. When HSF1 is activated, it binds the HSE to transcribe RFP reporter. (B) Full sequence of HSE. Green letters indicate HSF binding domains. The details of reporter evaluation have been described previously (Torii et al., 2017). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 2

HSE-RFP reporter induction in the spinal cord and cerebral cortex following spinal cord injury. (A–D) HSE-RFP reporter expression was detected by immunohistochemistry with 3,3'-Diaminobenzidine (black) in coronal (A and C, at the T9 level) and sagittal (B and D, lateral to the lesion) sections around the lesion site 4 days after spinal cord injury (C and D) and at the same regions in the sham control (A and B). HSE-RFP reporter expression was observed in cells surrounding the lesion site (C and D). Arrows in C and D indicates the regions with the reporter expression. Note that the center of section in C is damaged due to the lesion. The arrowhead in D indicates the level of the lesion. (E) Sagittal view of the brain with immunofluorescence labeling for Fluoro-Gold (FG, green) and RFP (red) 4 days after spinal cord injury. Strong reporter expression is observed in the FG⁺ backfilled corticospinal projection neurons in layer V (bracket) as well as cells in other layers in the M1 cortex. The inset shows a higher magnification view (from another sample) of FG⁺/RFP⁺ neurons in layer V and RFP⁺ neurons above/below layer V. (F) No reporter expression was observed in the M1 in sham brain labeled for RFP (red) and nuclei (DAPI stain, blue). M1: primary motor cortex, M2: secondary motor cortex, MPtA: medial parietal association cortex. Bars = 100 (A–D) and 200 (E and F) μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 3

HSE-RFP reporter induction in the spinal cord and dorsal root ganglia by sciatic nerve injury. (A-F”) Coronal sections of the spinal cord (A–D and F-F”: L2-3 levels, E and E’: T4-5 levels) and DRG (G and H) were labeled for indicated markers 3 days (A–D and F-F’”) or 2 weeks (E, E’ G and H) after sciatic nerve injury (SNI) or in sham controls. The color of letters for each marker corresponds to the color of the staining. The arrow in A’ indicates the WGA labeling in the ipsilateral ventral horn of the spinal cord. HSE-RFP reporter expression was observed in motor neurons in the ipsilateral ventral horn (A and C), sensory neurons in bilateral dorsal horns [A and D (contralateral side is shown)], contralateral tract in the ventrolateral region (E), and Iba1⁺ microglia in the intermediate gray matter bilaterally [arrows in F (ipsilateral side is shown)] within the spinal cord. E’ and F’, F” are higher magnification views of boxed areas in E and F, respectively. Reporter expression was also observed in ipsilateral (G) and contralateral (H) DRGs. Bars = 100 (A–B and E), 10 (C and D) and 20 (F, G and H) μm. (I) Schematic drawing illustrating the SNI model and reporter expression (red) in major neural circuits connected with the injured neurons in the spinal cord and DRG. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 4

HSE-RFP reporter induction in the red nucleus by sciatic nerve injury. (A–D) HSE-RFP reporter (red) and NeuN (green) labeling in red nuclei (RN) of the ipsilateral (A and C) and contralateral (B and D) sides 2-weeks after SNI (A and B) or in sham controls (C and D). Strong reporter expression was observed in neurons in RN of both sides in SNI mice (A and B), but not in sham controls (C and D). Bars = 50 μm. (E) Schematic drawing illustrating the reporter expression (red) in the bilateral RN in SNI model. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 5

Abnormal electrophysiological properties of WDR neurons in HSE-RFP reporter-expressing dorsal horns of the spinal cord after sciatic nerve injury. (A) (left) Schematic of in vivo electrophysiological recording of WDR neurons in the lumbar spinal segments (L2-3 levels) at 4 weeks after injury. A bracket indicates the target recording region in the laminae III-VI in the dorsal horn ipsilateral to the injury. (Right) Tissue section adjacent to the recording site, showing HSE-RFP reporter expression throughout the dorsal horn, with the highest in the superficial region. Broken lines demarcate the boundaries of different laminae (I–IV). Bar = 100 μm. (B–G) Spontaneous discharge of wide-dynamic range (WDR) neurons in the ipsilateral dorsal horn of the spinal cord at the lumbar level (see the detail in Experimental Procedures) in control (B–D) and the SNI (E–G) mice 4 weeks after injury. The results are displayed in waveforms and spike-frequency histograms recording for 30 s (B and E), individual waveform (C and F), and superimposed waveforms obtained in 1 s (D and G), indicating increased firing following sciatic nerve injury. (H) Firing frequency of WDR neurons in SNI mice is significantly higher than that in sham control mice. *p < 0.05 by Mann-Whitney U test (n = 16 neurons from 4 animals per group). Mean ± SD is presented.