Cyclosporine-A (CsA) is a potent immunosuppressive agent commonly used clinically after organ transplantation. However, CsA treatment induces numerous side effects in several organs such as nephrotoxicity, hypertension and hepatoxicity. It has been shown in vitro that CsA in mixed cell cultures of dorsal root ganglia and the central nervous system of 6-14-day-old chick embryos causes the strongest toxic effects in glia cells and fibroblasts. Recently, the interest of researchers has been focused on the double role played by CsA in the central and peripheral nervous systems. On the one hand, CsA produces pyramidal weakness, extrapyramidal syndrome and peripheral neuropathy but on the other hand, CsA has a neuroprotective effect against neurological insults and diseases. It has been suggested that neurotoxic effects of CsA may be caused by the lipophilic character of the drug leading to blood–brain barrier damage. Indeed, CsA inhibits P-glycoprotein function and reduces its expression through direct cytotoxicity to brain capillary endothelial cells and glial cells, increasing permeability and inducing apoptosis. In contrast, CsA prevents neuronal death in the nervous system inhibiting mitochondrial pore opening by interaction with cyclophillin and blocking nitric oxide synthase activity and N-methyl- -aspartate-induced release of glutamate. However, CsA does not inhibit production of reactive oxygen species, but reduces the drop in ATP levels and subsequent neuronal death. Most recently, it has been shown that acute administration of CsA induces antinociceptive effects reducing catalytic activity of neuronal nitric oxide synthase.

Moreover, CsA treatment results in a dose-related reduction of pain in an experimental model of neuritis suggesting that the drug, that modulates the immune system, may be useful for therapies for neuropathic pain. Other studies have demonstrated that immunosuppressants, such as CsA and FK506, have neurotrophic actions and markedly promote neurite extension and regeneration of dorsal root axons into the spinal cord in vivo. However, previous studies have not carefully examined the therapeutic efficacy of CsA on the peripheral nervous system.

The aim of the present study was to examine and evaluate the in vivo CsA effects on lumbar ganglion cells of rats with respect to expression of 2 proteins belonging to the Bcl-2 family, Bax and Bcl-2, which play an important role in apoptosis. Bax and Bcl-2 expression is a critical intracellular checkpoint of apoptosis within a distal common cell death pathway. When Bcl-2 is overexpressed in cells, it heterodimerizes with Bax and death is prevented (death antagonist). Overexpression of Bcl-2 promotes neuronal survival in vitro and in vivo. When Bax is overexpressed, apoptotic death in response to a death signal is accelerated. Therefore, Bax is designated as death agonist. So, the ratio of Bcl2/Bax is important for the determination of susceptibility to apoptosis. Indeed, this ratio in the cell governs whether the cell survives or dies.

Using an immunohistochemical method, we investigated: (1) expression of Bax and Bcl-2 proteins in lumbar ganglion cells; (2) the possible difference of Bcl-2 and Bax expression in neurons and satellite cells (SC); and (3) the relationship between Bcl-2 and Bax expression and effects of CsA on the peripheral nervous system.

For the experiments, 35 male Sprague-Dawley rats with an average weight of 150–180 g were used in accordance with national animal protection guidelines. The animals were divided into seven groups (each of five animals). Animals in group 1 were used as controls and received intrascapular castor oil injections daily. Groups 2, 4 and 6 were subcutaneously injected into the intrascapular area with 7 mg/kg/day of CsA in castor oil (Sandimmun; Sandoz, Basel, Switzerland; mean volume of each injection, 50 ?l) for 7, 14 and 21 days, respectively. Groups 3, 5 and 7 were subcutaneously injected into the intrascapular area with 15 mg/kg/day of CsA in castor oil (mean volume of each injection, 100 ?l) for 7, 14 and 21 days, respectively, according to.

At the end of each treatment, the animals were deeply anaesthetized with an intraperitoneal injection of 8% chloral hydrate and perfused with 10% buffered formalin. Ganglia were collected from the lumbar column, stored in fresh fixative and processed according to standard procedures, embedded in paraffin, and sectioned with a thickness of 5 ?m. Sections were treated immunohistochemically. Some sections were stained with hematoxylin and eosin (H&E).

According to, neuron types in ganglia were classified into three main populations: type A (large-sized and light neurons), B (medium-sized and dark neurons) and C (small-sized and dark neurons). Only neurons showing a nucleus in the section plane were included in the study.

Sections of lumbar ganglia were incubated in 3% H₂O₂ in methanol, for 20 min, washed in PBS (0.1 M, pH 7.4) for 10 min and then covered with normal goat serum (NGS) in a dilution of 1:5 for 15 min. Subsequently, sections were incubated overnight with polyclonal anti-Bcl-2 or anti-Bax antibodies (Santa Cruz Biotechnology, Santa Cruz CA, USA) in a humidified chamber at 4°C. After incubation, sections were then washed in PBS (10 min), incubated with biotinylated goat-anti-rabbit IgG (Dako, Milan, Italy), dilution 1:50, in the humidified chamber for 30 min, washed three times in PBS and incubated with avidin-biotin-horseradish peroxidase complex (ABC-kit; Dako), according to the manufacturer’s instructions, for 60 min at room temperature. The ABC complex was visualised by incubating the sections for 10–15 min in a stock solution of 1 mg/ml DAB in PBS with 150 ?l of a solution of 3% H₂O₂. Sections were dehydrated and mounted in mounting medium.

Staining intensity was graded as: +, when staining was faint or barely detectable; ++, when staining was moderately positive; +++, when staining was clearly positive; and ++++, when staining was strong. Two observers, unaware of the experiments, analysed 10 sections from each animal of all groups.

Negative control experiments were carried out by incubating the ganglia sections in PBS omitting the Bax and Bcl-2 antibodies. Specificity of antibody labelling was investigated using appropriate controls, by incubating tissue sections with non-immunized goat serum for Bax and Bcl-2 or PBS instead of the primary or secondary antibodies or ABC complex.

Different patterns of Bax and Bcl-2 expression were found in neurons and satellite cells (SC) of lumbar ganglia in control and CsA-treated animals. Results are summarized in Table 1 and Table 2.

The control ganglia sections incubated in the absence of anti-Bax and anti-Bcl-2 antibodies, did not show any immunostaining (data not shown).

Lumbar ganglia of untreated rats and rats treated with castor oil showed similar structural and immunohistochemical characteristics. Therefore, the data of these groups are described without distinction between them.

Neurons: Bax was faintly expressed in all types of neurons. Immunostaining appeared as granules scattered throughout the cytoplasm. Positivity was evaluated as weak.

Figure 1. Immunohistochemical staining of Bax in rat spinal ganglion cells of control rats (A) and after 21 days of CsA treatment (15 mg/kg daily; B). Arrow heads, satellite cells; open asterisk, type A neuron; close asterisk, type B neuron; arrow, type C neuron. Scale bar, 20 ?m.

SC: Bax immunostaining was granular and evident in the cytoplasm of SC. In fact, their cytoplasm showed many areas with moderate positivity and few areas with weak positivity.