Plasticity in expression of the glutamate transporters GLT-1 and GLAST in spinal dorsal horn glial cells following partial sciatic nerve ligation

Consistent with previous results [16, 17], the paw ipsilateral to the PSNL developed significant mechanical hypersensitivity as compared to the control side and to control rats. The paw withdrawal thresholds on the ipsilateral side were significantly decreased at 7 (4.6 ± 0.96 g) and 14 days (4.3 ± 1.12 g) after PSNL, compared with the control group (12.6 ± 3.84 g).

Glial fibrillary acidic protein (GFAP) immunoreactivity (IR) in the dorsal horn ipsilateral to the nerve injury showed a noticeable increase by 1 day after PSNL (Figure 1B) compared with the control group (Figure 1A). This became more pronounced by day 7 and 14 after injury (Figure 1C, Figure 1D).

The expression of GLT-1 and GLAST in the spinal dorsal horn in naïve animals were mainly expressed in the gray matter of the spinal cord, especially in the superficial laminae (Figure 2A, Figure 3A). Double staining results showed that both GLT-1 and GLAST were colocalized with the astrocytic marker GFAP (Figure 4D, 4E, 4F and Figure 5D, 5E, 5F), but not with the neuronal marker NeuN (Figure 4A, 4B, 4C and Figure 5A, 5B, 5C) or the microglia marker OX-42 (Figure 4G, 4H, 4I and Figure 5G, 5H, 5I).

The expression of GLT-1 was significantly decreased in the ipsilateral spinal dorsal horn at 7(p < 0.01) and 14 days (p < 0.01) after PSNL (Figure 2C, 2D) compared with controls (Figure 2A). Semiquantitative analyses showed that the relative intensity of GLT-1 decreased by 42 ± 15.79% at 7 days and 32.3 ± 13.13% at 14 days following injury, respectively, when compared to the control group (Figure 2E). Similarly, the expression of GLAST was reduced by 54 ± 22.13% at 7 days and by 42 ± 16.34% at 14 days after nerve injury (Figure 3C, 3D, 3E). Decreases in the expression of GLT-1 and GLAST were also found significant in a side to side comparison of the contralateral versus ipsilateral sides of the spinal cord within the PNSL animals (Figure 2E and Figure 3E). However, the total amount of GLT-1 and GLAST one day after ligation did not change. The results from the above suggest that the decrease in GLT-1 and GLAST, which was induced by PNSL, mainly occurred in astrocytes on the injured side.

Microglia in the spinal horn also showed evidence of activation due to the PSNL. An increase in OX-42-positive cells were noted by day 1 post PSNL (Figure 6B) compared with the control group (Figure 6A). The increase in OX-42 appeared to reach a maximum on day 7 after nerve injury (Figure 6C) and maintained this elevated level through day 14 after PSNL (Figure 6D).

As noted above, there was no co-localization of GLT-1 or GLAST with OX-42 positive microglia in the absence of nerve injury. By day 7 post PSNL double staining showed that OX-42-positive cells (Figure 4O) showed a de novo up-regulation of GLT-1that persisted through day 14 post PSNL (Figure 5S). Likewise, GLAST-positive OX-42 positive cells were observed at both days 7 (Figure 4O) and 14 post PNSL (Figure 4S). Quantitative analysis of the intensity of co-labeling for the glutamate transporters with GFAP and OX-42 over time is shown in Figure 7. Significant decreases in double label intensity for both GLT-1 and GLAST with GFAP were found at days 7 and 14 following PSNL. The opposite was found for GLT-1 and GLAST co-localization with OX-42 with both showing increases at days 7 and 14 following PNSL. Finally, no co-localization of either GLT-1 or GLAST was found in NeuN positive cells (neurons) at any time point (Figure 8D, E).

A total of 46 male Sprague-Dawley rats weighing 180–220 g were used. All experiments were conducted with the approval of the Institutional Animal Care and Use Committee at the M.D. Anderson Cancer Center and were in compliance with the National Institutes of Health Guidelines for Use and Care of Laboratory Animals. The minimum number of animals was used in each experiment, and in all cases every effort was made to minimize any pain or suffering in the subject animals.

Rats were anesthetized with 50 mg/kg (i.p.) of pentobarbital and the left sciatic nerve was exposed at the high thigh level. Partial sciatic nerve ligation was performed as previously described [16, 17]. Briefly, one-third to one-half of the left sciatic nerve was tightly ligated using 7-0 silk suture. Sham surgery in age matched animals consisted of exposing the left sciatic nerve but no ligation was made. The muscle layers were closed with 4-0 silk and the skin sealed with surgical clips.

The rats were accommodated to the testing environment by placement within testing chambers for 15–20 min on the three separate days just prior to the pre-operative testing. Mechanical sensitivity was assessed using von Frey hairs as described previously[35]. Briefly, rats were placed under separate transparent Plexiglas chambers positioned on a wire mesh floor. Fifteen minutes were allowed for habituation. Each stimulus consisted of a 2–3 s application of the von Frey hair to the middle of the plantar surface of the foot with 5 min interval between stimuli. Brisk withdrawal or licking of the paw following the stimulus was considered a positive response. The experimenter who conducted the behavioral tests was blinded to all treatments.

Rats were deeply anesthetized with urethane (1.5 g/kg, i.p.) at 1, 7 or 14 days following nerve injury, the chest opened, and then quickly perfused through the ascending aorta with warm heparinized saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2–7.4, 4°C. The L4–L5 spinal segments were removed and post-fixed for 3 h in the same fixative, and then stored in 30% sucrose overnight. Transverse sections (25 μm) were cut by cryostat and processed for immunohistochemical staining as previously described [36]. Sections were blocked with 3% donkey serum in 0.3% Triton X-100 for 1 hour at room temperature then incubated overnight at 4°C with guinea pig anti-GLT-1 antibody (1:2000, Chemicon) or rabbit anti-GLAST antibody (1:250, Abcam). The sections were then incubated for 1 h at room temperature with FITC-conjugated secondary antibody (1:250, Chemicon) or Cy³-conjugated secondary antibody (1:500, Chemicon). For double immunofluorescence, the spinal sections were incubated with a mixture of guinea anti-GLT1 antibody or rabbit anti-GLAST antibody and mouse anti-neuronal nuclei (NeuN, neuronal marker, 1:500, Chemicon), mouse anti-glial fibrillary acidic protein (GFAP, Astrocyte marker, 1:500, Chemicon) or mouse anti-OX-42 (Microglia marker, 1:500, Chemicon) antibody overnight at 4°C. Afterwards the sections were incubated with a mixture of FITC- and Cy3-conjugated secondary antibodies for 1 h at a room temperature. The stained sections were then examined with a Nikon E600 (Nikon Instech Co, Japan) fluorescence microscope and images were captured with a CCD spot camera.

The relative intensity of GLT-1-IR and GLAST-IR per section was measured in the spinal dorsal horn using a computerized image analysis system (NIS-Elements, BR 2.30). An optic threshold was set above background level firstly to identify positively stained structures. Relative intensity values used for comparison were calculated by multiplying the mean optic density in areas of interest and then subtracting out background staining (mean optic density * positive area – value of background). In each rat, four to six sections of the spinal cord at each time point were selected randomly. An average percentage of GLT-1-IR, GLT-1 + OX-42-IR, GLT-1 + GFAP-IR, GLAST-IR, GLAST + OX-42-IR, and GLAST + GFAP-IR relative to the control group, was obtained for each animal across different time points, and then the mean ± SE among the animals was determined. Four to eight rats were included for each group for quantification of the results. All measurements were performed by blinded evaluators. Data were compared with student's t-test or one-way ANOVA followed by Neuman-Keuls tests. P < 0.05 was considered significant.