Figure 1.
Effects of intrathecal administration of c-fos ASO (A) or L-AA (B) on SNL-induced mechanical allodynia.
A, SNL rats in saline and c-fos SO group demonstrated significant ipsilateral allodynia since 1 d after the surgery. Intrathecal administration of c-fos ASO (50 µg/10 µl, starting 4 h prior to the surgery and once a day for a further 3 d following surgery) significantly reversed the ipsilateral allodynia. The preventing effect of ASO weakened gradually but still significant 7 d after SNL B, Intrathecal administration of astroglial toxin L-AA (100 nmol/10 µl, injected daily from 1 d before till 3 d after the surgery) reversed mechanical allodynia only at the late stage. The data are presented as the mean ± S.E.M. * and ** indicate statistically significant difference with P<0.05 and P<0.01 compared to saline control of each time point, respectively.
Figure 2.
Immunofluorescent staining for Fos and GFAP in the spinal dorsal horn after SNL.
A–A', SNL induced a significant up-regulation of Fos expression in the ipsilateral dorsal horn 2 h after SNL. B–B', GFAP immunoreactive astrocytes were significantly activated 1 w after nerve injury in the ipsilateral compared to the contralateral dorsal horn. Scale bar = 200 µm.
Figure 3.
Double immunofluorescence for Fos (red)/NeuN (green) and Fos (red)/GFAP (green) in the ipsilateral dorsal horn 3 d after SNL.
A' and B' are the magnified images of the rectangles indicated in the above top panels in A and B, respectively. Double immunofluorescence indicated that few GFAP-IR astrocytes were Fos positive (shown by double arrowheads in B'), while most of them were nuclei of activated neurons (shown by double arrowheads in A'). These activated neurons and astrocytes were mainly distributed in the superficial layer of dorsal horn, and the Fos-positive neurons were surrounded with many GFAP-positive astrocytic processes formed close relationship with each other. The figures shows representative photomicrographs obtained from a minimum of three independent experiments. Fos and NeuN double labeled neurons observed in the ipsilateral superficial dorsal horn were counted and the proportion of double labeling neurons in total Fos positive cells were calculated. The statistical results of these comparisons are presented in C. Scale bar = 200 µm in A, B and 80 µm in A' and B'.
Figure 4.
The expressions of Fos and GFAP in the ipsilateral dorsal horn in saline, c-fos SO and ASO groups after SNL.
Compared to saline or c-fos SO treated group, c-fos ASO produced a significant inhibitory effect on Fos protein expression (A–A″), as well as on astrocytic activation (B–B″, C–C″) in the spinal dorsal horn. D, Statistics of the time course study. The baseline level of Fos and GFAP was set as 100%, respectively. The changing course of Fos (red curves) or GFAP (green curves) was determined with comparison to the relative baseline value. The relative increase in the number of Fos-positive neurons reached a peak 2 h in the SO and saline group, then fell back quickly to the normal level in 1 w. However, Fos expression was maintained at the baseline level by ASO treatment. The GFAP expression presented a different changing course tendency: increasing since day 3 and keeping a high level 1 w after SNL. Interestingly, the increase of GFAP expression was interfered with by ASO treatment on day 3 and 7. #, P<0.05 and # #, P<0.01, as compared to the baseline value; *, P<0.05 and **, P<0.01, as compared to the saline control in the same time point. Scale bar = 200 µm.
Figure 5.
The effects of L-AA on astrocytic activation and Fos expression in the ipsilateral dorsal horn induced by SNL.
Compared to the saline control group, L-AA produced a marked reduction of GFAP-positive astrocytes in the dorsal horn (A, A'). L-AA did not influence the numbers of NeuN-expressing neurons indicating the specific role of this astroglial toxin (B, B'). How ever, it did significantly suppress the Fos expression on POD 3 (C, C'). D, The changing course of astrocytic activation and Fos expression after L-AA administration to the SNL rats. The baseline level of Fos and GFAP was set as 100%, respectively. The changing course of Fos (red curves) or GFAP (green curves) was determined with comparison to the relative baseline value. L-AA postponed astrocytic activation from day 3 (saline control) to day 7. Meanwhile, this astroglial toxin shortened the duration of Fos expression from 7 d (saline control) to 3 d post SNL, although it did not change the peak level of Fos expression. #, P<0.05 and # #, P<0.01, as compared to the baseline value; *, P<0.05 and **, P<0.01, as compared to the saline control in the same time point. Scale bar = 200 µm.
Figure 6.
Schematic illustration of the positive feedback circuit between spinal neurons and astrocytes in neuropathic pain state.
After peripheral nerve injury, various transmitters like substance P (SP), excitatory amino acids (EAA), were released from the presynaptic terminals in the superficial dorsal horn, which caused immediate (in hours) neuronal activation in the spinal dorsal horn. Neuronal activation is followed by a higher degree of synthesis and release of excitatory transmitters, which triggered late (>3 d) astrocytic activation. Activated astrocytes also produced neuroexcitatory substances, like IL-1β, IL-6, NO, and so on. These neuroexcitatory substances could action back to modify neuronal behavior, contributing to pain facilitation.
Figure 7.
Black bar represents the period during which behavioral test was performed daily. Grey bar represents the period when drugs or saline was injected once a day. Stars represent the time points when rats were sacrificed for immunohistochemistry. Intrathecal intubation was performed on rats and followed by 4-day recovery. Behavioral test was performed 1 d before SNL to provide baseline threshold. Drugs and saline were injected 4 h prior to SNL and once a day for 3 more times after SNL. Behavioral test was performed once a day from 4 h post SNL till POD 7. SNL: spinal nerve ligation, POD: post-operation day.