Preserved acute pain and reduced neuropathic pain in mice lacking PKCγ

Science

Volumn 278, Issue 5336, 1997, Pages 279-283

  Malmberg, Annika B ^a   Chen, Chong ^b   Tonegawa, Susumu ^b   Basbaum, Allan I ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b HOWARD HUGHES MEDICAL INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ISOENZYME; PROTEIN KINASE C;

ARTICLE; CELL SUBPOPULATION; INTERNEURON; MOUSE; NERVE INJURY; NEUROCHEMISTRY; NONHUMAN; PAIN; PRIORITY JOURNAL; SPINAL CORD DORSAL HORN;

ANIMALS; GANGLIA, SPINAL; GENE DELETION; HYPERALGESIA; INFLAMMATION; INTERNEURONS; ISOENZYMES; LIGATION; MICE; MICE, KNOCKOUT; NEUROPEPTIDE Y; PAIN; PAIN THRESHOLD; PROTEIN KINASE C; RECEPTORS, NEUROKININ-1; SCIATIC NERVE; SIGNAL TRANSDUCTION; SPINAL CORD; SUBSTANCE P;

EID: 0030881680     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5336.279     Document Type: Article

References (44)

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14. Nerve injury was produced by tying a tight ligature around approximately one-third to one-half of the diameter of the sciatic nerve, similar to the approach described for rats by Seltzer and colleagues [Z. Seltzer, R. Dubner, Y. Shir, Pain 43, 205 (1990)]. The general appearance and motor function are normal in the injured mice; occasionally, the mice hold the ligated paw in a protected position when they are not moving. We have not observed autotomy. We assessed mechanical allodynia with von Frey hairs using the up-down paradigm [S. R. Chaplan, F. W. Bach, J. W. Pogrel, J. M. Chung, T. L. Yaksh, J. Neurosci. Methods 53, 55 (1994)], and thermal sensitivity by measuring paw withdrawal latencies to a radiant heat stimulus [K. Hargreaves, R. Dubner, F. Brown, C. Flores, J. Joris, Pain 32, 141 (1988)]. In previous studies we found that partial nerve injury produces a significant decrease in thermal latencies for 5 to 6 weeks and mechanical allodynia for more than 2 months. In all studies, the observer was blind to the genotype of the mice. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.
15. Nerve injury was produced by tying a tight ligature around approximately one-third to one-half of the diameter of the sciatic nerve, similar to the approach described for rats by Seltzer and colleagues [Z. Seltzer, R. Dubner, Y. Shir, Pain 43, 205 (1990)]. The general appearance and motor function are normal in the injured mice; occasionally, the mice hold the ligated paw in a protected position when they are not moving. We have not observed autotomy. We assessed mechanical allodynia with von Frey hairs using the up-down paradigm [S. R. Chaplan, F. W. Bach, J. W. Pogrel, J. M. Chung, T. L. Yaksh, J. Neurosci. Methods 53, 55 (1994)], and thermal sensitivity by measuring paw withdrawal latencies to a radiant heat stimulus [K. Hargreaves, R. Dubner, F. Brown, C. Flores, J. Joris, Pain 32, 141 (1988)]. In previous studies we found that partial nerve injury produces a significant decrease in thermal latencies for 5 to 6 weeks and mechanical allodynia for more than 2 months. In all studies, the observer was blind to the genotype of the mice. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.
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19. For immunocytochemistry, the mice were deeply anesthetized with pentobarbital and intracardially perfused with 0.1 M phosphate-buffered saline followed by 10% formalin. After the perfusion, the spinal cord, DRG, and superior cervical ganglion were removed and processed for immunocytochemistry and quantified as described [C. Abbadie, J. L. Brown, P. W. Mantyh, A. I. Basbaum, Neuroscience 70, 201 (1996)]. The spinal cord was sectioned transversely in 30-μm-thick sections on a freezing microtome. DRG and superior cervical ganglia were cut in 12-μm-thick sections on a cryostat. Fos protein immunoreactivity was examined on spinal cord sections from mice that were studied in the formalin test. SP, NK-1 receptor, and NPY immunostaining were performed on spinal cord sections from nerve-injured mice. Staining for the different PKC isozymes was done on tissue from noninjured mice. We used the following dilutions of antisera: 1:30,000 for Fos (provided by D. Slamon, University of California, Los Angeles), 1:30,000 to 1:60,000 for SP (Peninsula Laboratories), 1:20,000 for NK-1 receptor (provided by S. Vigna, University of North Carolina), 1:20,000 to 1:40,000 for NPY (Peninsula Laboratories), and 1:10,000 for PKCα, -βI, -βII, and -γ (Santa Cruz Biotechnology Laboratory).
20. H. Liu et al., Proc. Natl. Acad. Sci. U.S.A. 91, 1009 (1994); J. L. Brown et al., J. Comp. Neurol. 356, 327 (1995).
21. H. Liu et al., Proc. Natl. Acad. Sci. U.S.A. 91, 1009 (1994); J. L. Brown et al., J. Comp. Neurol. 356, 327 (1995).
22. Total nerve transection was performed in halothane-anesthetized mice by ligating and then transecting the sciatic nerve at the mid-thigh level. Fourteen days later, the mice were transcardially perfused with 10% formalin fixative, and the spinal cord and DRG were removed and prepared for immunocytochemistry (8). Spinal cord sections and DRG were stained for SP or NPY immunoreactivity. To determine the percentage of immunoreactive cells in the DRG, we counted all cell bodies (with visible nuclei) in six sections of the L4 and L5 ganglia from five wild-type and five mutant mice. We never recorded NPY immunoreactivity in mice without nerve injury or on the contralateral side in mice with nerve injury. In contrast, 14 days after nerve injury in wild-type mice, we found NPY immunoreactivity in 18 ± 2% of the L4 and L5 DRG cell bodies. In the mutant mice 16 ± 2% of DRG cell bodies contained NPY immunoreactivity; the differences were not statistically significant [P > 0.05, protected least significant difference (PLSD) Fisher's test]. The density of NPY labeling in the dorsal horn of wild-type mice also increased, to 124 ± 3%, comparing the injured to the uninjured sides (8). The mutant mice showed less of an increase (110 ± 3%) at the L4-L5 spinal segment; this difference was statistically significant (P < 0.05, PLSD Fisher's test). SP immunoreactivity of the DRG was decreased on the injured sides in both wild-type and mutant mice (9 ± 1% and 8 ± 1% of all DRG cells were SP-immunoreactive); these differences were not significantly different, but both were different from the immunoreactivity of the respective non-injured sides (18 ± 1% and 19 ± 1% of all DRG cells were SP-immunoreactive). In the spinal cord dorsal horn, nerve transection in wild-type mice produced a 35 ± 3% decrease in SP immunoreactivity compared with the noninjured side. In contrast, the mutant mice only showed a 14 ± 4% reduction in dorsal horn SP immunoreactivity on the injured side. The difference was significant (P < 0.01, PLSD Fisher's test).
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25. Four wild-type and four mutant mice received a 10-μl intraplantar injection of 2% formalin solution, and the amount of time that the mice licked the injected paw was monitored for 60 min. The incidence of licking was measured in 2-min periods at 5-min intervals. To quantify the magnitude of the inflammatory response, we measured the paw diameter with a spring-loaded caliper (Mitutoyo) 90 min after the formalin injection. There was no difference in licking behavior during the first phase of the formalin test between wild-type (52 ± 6 s) and mutant (41 ± 5 s) mice (P > 0.05, t test). In contrast, the second phase of the formalin test was significantly reduced (P < 0.05, t test) in mutant (39 ± 10 s) compared with wild-type mice (90 ± 12 s). The formalin-evoked paw edema was also significantly less (P < 0.01, t test) in mutant (2.61 ± 0.08 mm) compared with wild-type mice (3.30 ± 0.09 mm).
26. The mice were perfused 90 min after the formalin test, and the spinal cord tissue was prepared for immunocytochemistry as described in (8). To quantitate the number of Fos-like immunoreactive neurons, we took photographs of 5 to 10 sections from the L4-L5 spinal segment at low (×4) power on a Nikon Microphot-FXA microscope. The photographs were divided into four segments: laminae I-II, II-VI, V-VI, and the ventral horn. A person blinded to the groups counted the number of Fos-immunoreactive neurons. Five to 10 spinal cord sections were counted per mouse and averaged so that each mouse had a mean value for regional Fos immunoreactivity. There was a significant difference in Fos expression between the two groups (P < 0.05, two-way ANOVA comparing group and laminae). Specifically, the mutant mice showed a significant reduction (P < 0.05, PLSD Fisher's test) of Fos-positive cells in laminae I and II compared with wild-type mice (41 ± 3 versus 60 ± 4 cells).
27. To study capsaicin-induced plasma extravasation, we anesthetized five mice from each group with 50 mg of pentobarbital per kilogram of body weight and made an intravenous injection of Evan's Blue (10 mg/kg) into a tail vein. Five minutes later, 0.1 μ9 of capsaicin (8-methyl-N-vanillyl-6-noneaneamide; Sigma, St. Louis, MO) in 5 μl of vehicle (10% ethanol, 10% Tween-80, and 80% saline) or vehicle alone was injected intradermally into the dorsal part of the hindpaw, and punches of skin were sampled 30 min later. The Evan's Blue was extracted from the tissue samples by incubation in formamide; extravasated protein was measured spectrophotometrically as described (21). In wild-type mice Evan's Blue was extracted from the paw skin at a concentration of 1.1 ± 0.1 μg/ml. Mutant mice showed significantly less (P < 0.05, t test) extravasated Evan's Blue (0.6 ± 0.1 μg/ml).
28. 4 and stained in 2% aqueous uranyl acetate for 30 min. The dorsal roots were then dehydrated in ascending concentrations of ethanol, passed into propylene oxide, and embedded in Durcupan resin (Fluka). Finally, the ultrathin sections were placed on single-hole grids coated with butvar and stained with uranyl acetate and lead citrate. The thin sections of the dorsal roots were examined with the electron microscope, and photographs were taken at an original magnification of ×1600. Myelinated and unmyelinated axons from the dorsal roots were counted from montages of electron micrographs. Neither the morphology nor the numbers of myelinated or unmyelinated axons differed between mutant and wild-type mice. Specifically, the number of myelinated fibers was 2542 and 2854 in the two wild-type mice and 2791 and 2712 in the two mutant mice. The number of unmyelinated fibers was 5065 and 5648 in the wild-type mice and 5360 and 5585 in the mutant mice.
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30. Animals were prepared as described in (8). Dense PKCγ immunoreactivity was found in lamina IIi and the dorsal corticospinal tract in the spinal cord of wild-type mice. This expression pattern is in agreement with a previous study [M. Mori, A. Kose, T. Tsujino, C. Tanaka, J. Comp. Neurol. 299, 167 (1990)]. No PKCγ expression was found in either the DRG or the superior cervical ganglion. There was no PKCγ immunoreactivity in the spinal cord or brain of the mutant mice.
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44. We thank A. Doupe for her critical comments and H. Liu and H. Wang for help with electron microscopic analysis of the dorsal roots. Supported by NIH grants DA08377, NS 14627, and NS 21445 (to A.I.B.), the Swedish Cancer Foundation (to A.B.M.), and the Pharmaceutical Research and Manufacturers of America Foundation (to A.B.M.). • SCIENCE • VOL. 278 • 10 OCTOBER 1997