Endoplasmic reticulum stress impairment in the spinal dorsal horn of a neuropathic pain model methods
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Intracellular ATF6 location in the nuclei of neurons of the dorsal horn following SNL
Among UPR sensor pathways involving PERK, IRE1-XBP1, and ATF6, we first examined the translocation of ATF6 using an immunofluorescence assay. Proteolytic activation of ATF6 in the ER stress response is induced by membrane-bound factors, and this process has been termed "regulated intra-membrane proteolysis" (RIP). The cytoplasmic fragments of ATF6 move to the nucleus and activate the expression of ER stress target genes by binding to the ER stress response element (ERSE) within their promoter regions. Our results showed an upregulation of ATF6 induced by L5 SNL in the ipsilateral lumbar spinal dorsal horn when compared to the contralateral ( p < 0.05) and the sham groups ( p < 0.001; ). Double immunofluorescence staining was performed to assess the localization of ATF6 IR cells using DAPI, a general nuclear marker, in the superficial dorsal horn...
Increased ER swelling in dorsal horn neurons after SNL
Next, we used electron microscopy to evaluate ultrastructural changes in the ER 14 days after SNL. Neurons in the sham spinal cord appeared to be normal, with relatively healthy-looking ER, mitochondria, and nuclei ( ). Fourteen days after SNL, the mitochondria and the nuclei in the neurons also appeared normal with no appreciable pathological changes; however, several swollen ER lumen were seen, together with mild protein aggregation ( ). These data suggest that ER stress-induced ER cisternae swelling in the ipsilateral spinal dorsal horn after SNL.
Inhibition of the ATF6 pathway counteracts SNL-induced neuropathic pain
However, CatWalk XT gait analysis indicated that the Print Area and Stand Phase differed significantly according to the ATF6 siRNA injection ( ). The Print Area is basically at least as large as Max Contact Area and shows the ratio of max contact area of contralateral and ipsilateral paw (total percentage of contra- and ipsilateral paw area is 100%). Before SNL, the ratio of contralateral and ipsilateral side was not different. Five days after the surgery, the Print Area of the two ipsilateral paw groups were significantly reduced to 22.60% and 25.58% (both p < 0.001 compared to contralateral paw values) at 30 min prior to vehicle and ATF6 siRNA injection, respectively. Print Area in ipsilateral paw recovered from 25.58% to 40.98% compared to contralateral paw in 3, 5, 9 days after ATF6 siRNA injection ( ). Before SNL, the duration of the Stance Phase was 50.46% and 50.43...
Electron microscopic examination
Representative photomicrographs of samples were taken from three rats in each group following SNL or sham surgery. After 14 days from the surgery, the rats were perfused with pre-cooled phosphate buffered saline solution (PBS, pH 7.4) followed by 2.5% glutaldehyde, 1% PFA, 0.1% Picric acid in 0.1 M PB after anesthetization. The lumbar enlargement (L4-L6) regions of the spinal cords were removed and kept in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4). Ipsilateral dorsal horn of rat was selected for analysis. The selected areas were processed by post fixation in 1% osmium tetroxide for 1 h, dehydrated in graded ethanol and embedded in epoxy resin. Polymerization was performed at 80 °C for 24 h. Blocks were cut on a Reichert ultramicrotome into ultrathin sections (60 mm), which were post-stained with uranyl acetate and lead citrate, and viewed...
Materials and Methods
Male Sprague-Dawley rats (180-200 g, Koatech, Korea) were housed individually in cages on a standard 12:12 h light:dark cycle. Water and food were provided to the rats ad libitum. Rats were transported to the laboratory approximately 1 h prior to each experiment. All experiments were carried out with the approval of the Animal Care and Use Committee at the Chungnam National University (CNU-00491) and were accordance with the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain. The same method and procedure for surgery were used as a previously published paper.
Pain threshold assessment
Mechanical paw withdrawal thresholds (PWTs) were measured using the up-down Von Frey testing following spinal nerve ligation or sham surgery. The same methods and procedure were used as a previously published paper.
RNAi administration
Neuropathic pain was induced in rats by SNL surgery. The rats were divided into siRNA (Rn-Atf6-predicted-1, SI04729928, Qiagen) and vehicle groups, with at least six rats per group. siRNA and vehicle doses were prepared immediately just before administrating the RNA solution (200 µM in annealing buffer) with a transfection reagent, (Invitrogen, #11668-019), at a 1:4 ratio (w:v). At this ratio, the final concentration of RNA as an RNA/lipid complex was 2 µg in 10 µL. Then, 10 µL of siRNA or Lipofectamine with scrambled siRNA (defined as vehicle) were delivered to the lumbar region of the spinal cord via intrathecal (IT) injection. Injections were given daily for three consecutive days. Nociceptive testing and tissue collection were carried out at days after SNL.
Measurement outputs
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Gait recovery and locomotor function
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- Per-run gait capture with paw placement, timing, and stride features for each animal
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- Cleaned gait metrics table and recovery trend summary across timepoints
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- Group comparisons of gait indices, stride metrics, or recovery curves
Microscopy-based tissue or cell-state imaging
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Evidence quotes (7)
Among UPR sensor pathways involving PERK, IRE1-XBP1, and ATF6, we first examined the translocation of ATF6 using an immunofluorescence assay. Proteolytic activation of ATF6 in the ER stress response is induced by membrane-bound factors, and this process has been termed "regulated intra-membrane proteolysis" (RIP). The cytoplasmic fragments of ATF6 move to the nucleus and activate the expression of ER stress target genes by binding to the ER stress response element (ERSE) within their promoter regions. Our results showed an upregulation of ATF6 induced by L5 SNL in the ipsilateral lumbar spinal dorsal horn when compared to the contralateral ( p < 0.05) and the sham groups ( p < 0.001; ). Double immunofluorescence staining was performed to assess the localization of ATF6 IR cells using DAPI, a general nuclear marker, in the superficial dorsal horn. While normally the ATF6 IR area was located in the cytoplasm, the positive fluorescence area was more visible within the nucleus in the ipsilateral spinal dorsal horn when neuropathic pain was induced for 14 days ( ). We also found ATF6 IR cells colocalized with NeuN in the superficial dorsal horn...
Next, we used electron microscopy to evaluate ultrastructural changes in the ER 14 days after SNL. Neurons in the sham spinal cord appeared to be normal, with relatively healthy-looking ER, mitochondria, and nuclei ( ). Fourteen days after SNL, the mitochondria and the nuclei in the neurons also appeared normal with no appreciable pathological changes; however, several swollen ER lumen were seen, together with mild protein aggregation ( ). These data suggest that ER stress-induced ER cisternae swelling in the ipsilateral spinal dorsal horn after SNL.
However, CatWalk XT gait analysis indicated that the Print Area and Stand Phase differed significantly according to the ATF6 siRNA injection ( ). The Print Area is basically at least as large as Max Contact Area and shows the ratio of max contact area of contralateral and ipsilateral paw (total percentage of contra- and ipsilateral paw area is 100%). Before SNL, the ratio of contralateral and ipsilateral side was not different. Five days after the surgery, the Print Area of the two ipsilateral paw groups were significantly reduced to 22.60% and 25.58% (both p < 0.001 compared to contralateral paw values) at 30 min prior to vehicle and ATF6 siRNA injection, respectively. Print Area in ipsilateral paw recovered from 25.58% to 40.98% compared to contralateral paw in 3, 5, 9 days after ATF6 siRNA injection ( ). Before SNL, the duration of the Stance Phase was 50.46% and 50.43% in control and ATF6 siRNA injection groups, respectively. Five days after the surgery, the duration of the Stance Phase of the ipsilateral paw was significantly reduced to 15.56% ( p < 0.001) and 27.75% ( p < 0.001) compared to contralateral paw values, respectively, at 3...
Representative photomicrographs of samples were taken from three rats in each group following SNL or sham surgery. After 14 days from the surgery, the rats were perfused with pre-cooled phosphate buffered saline solution (PBS, pH 7.4) followed by 2.5% glutaldehyde, 1% PFA, 0.1% Picric acid in 0.1 M PB after anesthetization. The lumbar enlargement (L4-L6) regions of the spinal cords were removed and kept in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4). Ipsilateral dorsal horn of rat was selected for analysis. The selected areas were processed by post fixation in 1% osmium tetroxide for 1 h, dehydrated in graded ethanol and embedded in epoxy resin. Polymerization was performed at 80 °C for 24 h. Blocks were cut on a Reichert ultramicrotome into ultrathin sections (60 mm), which were post-stained with uranyl acetate and lead citrate, and viewed under a Hitachi 7100 electron microscopy.
Male Sprague-Dawley rats (180-200 g, Koatech, Korea) were housed individually in cages on a standard 12:12 h light:dark cycle. Water and food were provided to the rats ad libitum. Rats were transported to the laboratory approximately 1 h prior to each experiment. All experiments were carried out with the approval of the Animal Care and Use Committee at the Chungnam National University (CNU-00491) and were accordance with the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain. The same method and procedure for surgery were used as a previously published paper.
Mechanical paw withdrawal thresholds (PWTs) were measured using the up-down Von Frey testing following spinal nerve ligation or sham surgery. The same methods and procedure were used as a previously published paper.
Neuropathic pain was induced in rats by SNL surgery. The rats were divided into siRNA (Rn-Atf6-predicted-1, SI04729928, Qiagen) and vehicle groups, with at least six rats per group. siRNA and vehicle doses were prepared immediately just before administrating the RNA solution (200 µM in annealing buffer) with a transfection reagent, (Invitrogen, #11668-019), at a 1:4 ratio (w:v). At this ratio, the final concentration of RNA as an RNA/lipid complex was 2 µg in 10 µL. Then, 10 µL of siRNA or Lipofectamine with scrambled siRNA (defined as vehicle) were delivered to the lumbar region of the spinal cord via intrathecal (IT) injection. Injections were given daily for three consecutive days. Nociceptive testing and tissue collection were carried out at days after SNL.
Machine-readable layer
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"text": "Among UPR sensor pathways involving PERK, IRE1-XBP1, and ATF6, we first examined the translocation of ATF6 using an immunofluorescence assay. Proteolytic activation of ATF6 in the ER stress response is induced by membrane-bound factors, and this process has been termed \"regulated intra-membrane proteolysis\" (RIP). The cytoplasmic fragments of ATF6 move to the nucleus and activate the expression of ER stress target genes by binding to the ER stress response element (ERSE) within their promoter regions. Our results showed an upregulation of ATF6 induced by L5 SNL in the ipsilateral lumbar spinal dorsal horn when compared to the contralateral ( p < 0.05) and the sham groups ( p < 0.001; ). Double immunofluorescence staining was performed to assess the localization of ATF6 IR cells using DAPI, a general nuclear marker, in the superficial dorsal horn..."
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"text": "Next, we used electron microscopy to evaluate ultrastructural changes in the ER 14 days after SNL. Neurons in the sham spinal cord appeared to be normal, with relatively healthy-looking ER, mitochondria, and nuclei ( ). Fourteen days after SNL, the mitochondria and the nuclei in the neurons also appeared normal with no appreciable pathological changes; however, several swollen ER lumen were seen, together with mild protein aggregation ( ). These data suggest that ER stress-induced ER cisternae swelling in the ipsilateral spinal dorsal horn after SNL."
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