In unlesioned adult rats CATS- and CATX-immunoreactivities were found in cells of both grey and white matters (Fig. 1) throughout the entire length of the spinal cord. Most CATS-immunopositive cells were of small size and distributed uniformely (Fig. 1A, C, E, F). While CATS-immunopositive neurons were rare, CATX-immunoreactivity was found in nearly all neurons (Fig. 1B, D, H) and only in few small cells (Fig. 1D, J). The immunoreactivities were associated with spherical granules within the cytoplasm of cells, sparing the nucleus (Fig. 1E, D, H).

The first changes of CATS- and CATX-immunoreactivities were already notable 1 d after L5T. For both proteins we observed upregulations that were restricted to the ipsilateral fasciculus gracilis, the dorsal horn and layer IX in the ventral horn in the lumbar segment (Figs. 2 and 3). Within these regions, the number of small CATS- as well as CATX-immunopositive cells increased substantially (Fig. 2). Moreover, we found a numerical increase in CATS-immunopositive neurons, while the number of CATX-immunopositive neurons was constant. Interestingly, numerous small CATS as well as CATX-immunopositive cells engulfed large motoneurons in the ventral horn (Fig. 2G, I). Within the following days the ipsilateral increase in immunopositive cells in the fasciculus gracilis spread caudocranially to the upper SC segments and reached the gracile nucleus at 1 w after injury (Figs. 2L, N and 3). At that time-point this nucleus exhibited morphological signs of degeneration (data not shown).

We next demonstrated that the changes in CATS- and CATX-immunoreactivities are reflected by changes in the levels of the respective proteins and, above all, that these are also reflected by a change of activity. Therefore, we first analyzed the protein levels of CATX and CATS in the spinal cord of sham versus L5T animals at 8 d after injury, a time point when the increase in immunoreactivities in the spinal cord was at its maximum (Fig. 3).

Western blot analysis (n = 5 per group) revealed the proforms of CATS (37 kD) and CATX (34 kD), as the most prominent bands, while the prepro- and mature forms were below the detection level. The proforms of both enzymes were detected in all segments analyzed (L, lumbar; T, thoracic; C, cervical) of the adult rat spinal cord (Fig. 4A). The L5 nerve transsection produced an upregulation in all SC segments for CATS as well as CATX (Fig. 4A). The strongest increase in protein content was found in the T segment for both enzymes (CATS 63.6%; CATX 87.4%), while the increase in the L and C-segments was substantial (34–61.5%) but lower than in the T segment (Fig. 4A). Moreover, our Western blot analysis showed that in all SC segments the level of CATX is more than twice as high as the level of CATS. These results were confirmed in a second experiment with 4 animals per group (see Additional files 1 and 2).

This data is supported by measurements of CATX activity. CATS activity assays were not performed since the assay suffered from the lack of a specific substrate and a specific commercially available inhibitor for the enzyme, leaving doubts about the specificity of the assay in complex protein mixtures. At 8 d after L5T CATX activity increased strongly and highly significant (p ≤ 0.001) in the lumbar segment (Fig. 4B; 59% compared to sham, 90% compared to naive). CATB activity was not significantly changed (data not shown).

Concurrent with the upregulation of CATS and CATX a strong gliosis appeared in the affected regions. In the ipsilateral fasciculus gracilis we observed numerous ED1-immunopositive macrophages (Fig. 5A, B), while in the ipsilateral DH and VH PT66-immunopositive microglia and GFAP-immunopositive astrocytes were more abundant than in the contralateral side (Fig. 5C–H).

To determine the phenotype of CATS and CATX cells in vivo we performed double immunofluorescence. The majority of CATS-immunopositive cells expressed the microglia-marker PT66 or the astrocyte-marker GFAP (Fig. 6A'–A"'), while only a small number of neurons were CATS-immunopositive (Fig. 6E). In contrast, CATX-immunoreactivity colocalizes only with single glial cells (Fig. 6B'–B"') but was more abundant in neurons (Fig. 6D). All ED1-immunopositive macrophages expressed both proteins (Fig. 6C'–C"').

Male Wistar rats (Janvier, Le Genest Saint Isle, France) with a weight of 200–250 g were used. Animals were housed in a climate-controlled room on a 12–12 light-dark cycle. Food and water were available ad libitum. All procedures were approved by the local animal usage committees according to German guidelines on animal care and use.

Prior to the operation, rats were deeply anesthetized with pentobarbitone at a dose of 50 mg/kg i.p. The L5T model was achieved by transection of the left L5 spinal nerve in a procedure modified from Kim and Chung 4748. In sham controls the sciatic nerve was exposed but not transected.

Rats were sacrificed at day 1 (1 d) – 35 d for Western Blot analysis (L5T n = 5, sham n = 5), immunohistochemistry (L5T n = 2–4, sham n = 4) and activity assays (L5T n = 4, sham n = 7, naïve n = 4).

Withdrawal tests for evaluation of tactile allodynia were measured by the use of the dynamic plantar aesthesiometer. The animals were placed into raised plexiglass boxes with mesh flooring and allowed to acclimatize for at least 15 min until exploratory behavior ceased. Sampling was conducted by a metal filament which was applied manually to the ventral mid-plantar hind paw. The force raised (0–50 g) with time (20 s) until the rat lifted its paw. The mean withdrawal threshold for both hind paws was taken from a set of three applications, not less than 2 min apart.

For immunoblotting, spinal cord and brain tissues were homogenized in triple detergent lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, complete protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) using a Teflon/glass homogenizer at 4°C. Homogenized samples were kept on ice for 30 min und subsequently centrifuged for 10 min in a precooled centrifuge at 12,000 g. The supernatant was collected and subsequently diluted 1:1 in 2× Laemmli sample buffer and boiled for 5 min. Protein determination was performed by the method of Neuhoff and coworkers 49.

For immunohistochemistry, animals were transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). The brain, the spinal cord and the ipsi- and contralateral nerves L4–L6 were excised and postfixed for 24 h in the perfusion fixative. Spinal cords were subdivided into the spinal cord segments according to the numbers of the related spinal nerves and all segments and the L5 (ipsi- and contralateral) nerves were embedded in paraffin. Serial, transversal 18-μm-thick sections were cut throughout all spinal cord segments, the hindbrain and the peripheral nerves. Sections were mounted on Superfrost slides (Carl Roth, Karlsruhe, Germany).

For CATX activity assays, the tissues were thawed separately on ice and homogenized in 7-fold volume (w/v) of 100 mM NaCl, 50 mM NaOAc, 4 mM EDTA-Na₂, 0.1% Triton X-100, pH 5.0 50. All procedures were carried out at 4°C. After incubating the samples for 60 min on ice, they were centrifuged (60 min at 13,000 g) for elimination of debris. Supernatants were stored in aliquots at -80°C until further use. Protein contents of the preparations were measured by the method of Neuhoff 49.

Proteins were electrophoretically separated on a 10% polyacrylamide gel containing SDS and transferred onto a PVDF-membrane (Carl Roth, Karlsruhe, Germany) at 4°C with 200 mA for 1.5 h. Blocking was performed with 1.5% milk powder and 1% BSA in TBS-Tween (0.1% Tween, 20 mM TBS) at RT for 1 h. Incubation with the primary antibodies goat anti-mouse CATX (1:500; R&D Systems, Wiesbaden, Germany) or goat anti-human CATS (1:200; R&D Systems) was conducted in blocking buffer overnight at 4°C. The next day blots were incubated in HRP-coupled anti-goat (1:50,000 in 1.5% milk powder in TBS-Tween; Amersham Biosci., München, Germany) at RT for 1.5 h, followed by detection with the ECL-Plus system (Amersham Biosci.).

To control protein loading, the blots were incubated with mouse anti-chick α-tubulin (1:400,000; Sigma, Deisenhofen, Germany) followed by HRP-coupled anti-mouse (1:50,000 in 1.5% milk powder in TBS-Tween; Amersham Biosci.) and the ECL-Plus system as described above.

Spinal cord tissues were used for CATX enzyme activity tests. CATX activity was measured in 25 mM CH₃COONa/1 mM EDTA/5 mM DTT (pH 3.5) with 10 μM MCA-R-P-P-G-F-S-A-F-K(Dnp)-OH (R&D Systems) as substrate. In parallel assays, the specific CATB inhibitor CA-074 (1 μM, Bachem, Weil am Rhein, Germany; Ki = 2 nM for purified rat CATB and 40–200 μM for CATH and CATL; 52) and the non-specific cysteine protease inhibitor E-64 (5 μM, Sigma) were added in order to distinguish between CATB activity and the entire cysteine protease activity. A clear determination of CATB activity is essential as CATB is also able to hydrolyze the substrate at these conditions. Activity of other proteases being able to cleave MCA-R-P-P-G-F-S-A-F-K(Dnp)-OH, like CATL and ECE-1, was undetectable (data not shown). Assays were performed at RT in black 96 multiwell plates (Falcon) in a total volume of 50 μl. Prior starting the assay by addition of substrate, the enzyme solutions were pre-incubated for 5 min with the different inhibitors or diluent at RT. After a 30 min incubation time the assays were stopped by the addition of 50 μl stop solution (100 mM CH₂ClCOOH, 70 mM CH₃COOH, 30 mM CH₃COONa, pH 4.3) and measured at 340/430 nm, according to the procedures described by 53. The activities of the proteases were calculated on the basis of relative fluorescent units. The total cathepsin activity was assessed by taking the difference between non-inhibited and E-64 inhibited samples. CATB activity corresponded to the part of the response that could be inhibited by CA-074. The difference between the fluorescence measured in the presence of the CATB inhibitor and the fluorescence measured in the presence of E-64 was attributed to CATX activity.