Introduction

Diabetes mellitus is a major health concern for human and according to the reports published by World Health Organization (WHO), the prevalence of diabetes for all age-groups worldwide is estimated to be 4.4% in 2030 (1). Diabetic neuropathies are heterogeneous groups of disorders which present a wide range of abnormalities. Such disorders are among the most common long-term complications of diabetes and are important sources of morbidity and mortality (2).

Almost 50% of the diabetic patients develop neuropathy with symptoms including spontaneous pain, hypoesthesia, allodynia and hyperalgesia (3). Despite significant progress in the treatment of pain, a variety of pathological pains, particularly diabetic neuropathic pain, cannot still be alleviated and they are refractory to the currently available analgesics. Therefore, biomedical investigations are performed
to reveal cellular and molecular factors that are affected in the central nervous system during chronic pain to introduce potential targets for new drugs.

Although hyperglycemia is considered to be a major pathogenic factor in the development of diabetic neuropathy, the mechanisms associated with this disease have not yet been fully understood (4).

It has been demonstrated that changes in the extracellular GABA levels and impaired GABA_A receptor-mediated inhibition are occurred in neuropathic situations (5, 6). Spinal delivery of bicuculline, a GABA_A receptor antagonist reduced formalin-evoked flinching and tactile allodynia in diabetic rats (7). Furthermore, GABA_A receptor-mediated depression of the spinal Hoffmann's reflex (a reflectory reaction of muscles after electrical stimulation of sensory fibers) was also absent in the  spinal cord of diabetic rats (7). It has been reported

that GABA content as well as GABA receptor binding sites significantly decreased in the brain stem of pancreatectomised hyperglycemic rats (8).

Neurosteroids are synthesized in oligodendro-cytes, astrocytes, schwann cells and some neurons, such as purkinje, retinal amacrine and ganglion cells and hippocampal neurons (9). Recent experimental findings bring new insights into the decline of neurosteroid synthesis in the CNS during aging and also in various neurodegenerative diseases (10).

Synthesis of neurosteroids declines during stressful conditions and in chronic inflammatory and neurodegenerative diseases. However, recent bio-chemical reports indicate that spinal neurosteroids are important factors for the control of pain (11) and also they show the potential applications of neurosteroids in the therapeutic management of painful neuropathic conditions (12, 13). It has been shown that some neurosteroids produce neuroprote-ctive effects against streptozotocin (STZ)-induced diabetic neuropathy at the neurophysiological, neur-opathological, biochemical and functional levels (14).

Since neurosteroids had shown protective effects in numerous cellular models of neurodegenerative diseases (9, 10) and elicited antinociception in neuropathic situations (11), the present study was designed to evaluate the preventive effects of neurosteroid allopregnanolone in animal models of diabetic neuropathic pain by evaluating its possible effect on the predicted diabetes-induced spinal GABA_A down-regulation.

Materials and Methods

Animals

The experiments were carried out on male Wistar rats that were housed in a room with 12 hr light/dark cycle with controlled temperature (22 ± 1°C). Food and water were available ad libitum. Animals (weighing 220-250 g) were handled daily (between 9:00-11:00 AM) 5 days before the experiment in order to adapt them to the experiment procedure and minimize nonspecific stress responses. Rats were randomly divided into several experimental groups, each containing 6-8 animals. The experiments followed the guidelines on ethics standard for investigation of experimental pain in animals (15) and approved by the Animal Experimentation Ethics Committee of Kerman Neuroscience Research Center (EC/KNRC/90).

In vivo experimental protocol

Diabetes was induced by a single intraperitoneal injection of 55 mg/kg (IP) streptozotocin (STZ) freshly dissolved in 0.1 mol/l citrate buffer. Rats receiving an injection of citrate buffer were used as control. One week later, diabetes was confirmed in STZ -injected rats by measuring serum glucose concentrations (16). The glucose concentration was assayed enzymatically using a glucose oxidase peroxidase (GOD-POD) kit (Pars Azmon Co, Iran). The rats with blood glucose ≥ 350 mg/dl were considered to have diabetes. One week after the STZ injection, the diabetic rats were given allopregnanolone (5 and 20 mg/kg/day for 6 weeks, orally). Seven weeks after the STZ administration, the animals were decapitated under CO₂ anesthesia. Control rats were killed in the same way 7 weeks after receiving vehicle injection.

Nociception assay

Nociceptive threshold was assessed by tail-flick test (17). The intensity of the beam was adjusted to produce a mean control reaction time between 4 and 6 sec. The cut-off time was fixed at 15 sec in order to avoid any damage to the tail. In this manner, we were able to reveal potential, subtle alternations that may occur in basal thermal nociception (18, 19). The         tail-flick latency for each rat was determined three times and the mean was designated as the baseline latency value. Experimentally-induced decreases in control tail-flick latency provide an indication of hyperalgesia as a marker of neuropathy.

Tissue extraction and preparation

Rats were anesthetized (using CO₂) and decapitated. The spinal column was cut through the pelvic girdle and a hydraulic extrusion was performed by inserting a 16 gauge needle into the sacral vertebral canal and expelling with ice-cold saline. The tissues were placed on ice in a glass petri dish, and the dorsal half of the lumbar spinal cord was dissected. Tissue samples were weighed and immediately frozen in liquid nitrogen and stored at -70°C until assay.

Rota-rod treadmill

Rota-rod treadmill test was performed to evaluate motor coordination of the animals (20). Briefly, the rats were placed individually on the rotating rod apparatus for two trials; on day zero and in the fourth week of the test. The rats were initially trained to maintain themselves on the rotating rod for more than 3 min. The rats were scored for their latency to fall (in seconds) in each trial.

RT-PCR analysis of GABA_A receptor

Total cellular RNAs was isolated from the dorsal half of the lumbar spinal cord by a modification of the guanidine isothiocyanate-phenol-chloroform method using RNX⁺ reagent and a semiquantitative RT-PCR method was used (21). Briefly, the RT-PCR reaction was performed using Oligo-dT primer and M-MuLV reverse transcriptase, based on the manufacturer protocol (Fermentas GMBH, Germany). The reactions were incubated at 42°C for 60 min and then inactivated at 70°C for 10 min. Three separate PCR reactions were used for studying the gene expression in the samples obtained from each rat.

Table 1. Effect of allopregnanolone (ALLO) on serum glucose levels and body weight of diabetic animals. Values represent mean ± SEM (n=6-8)

Each PCR reaction was carried out using selective forward and reverse primers for β-actin (as an internal standard) and γ2 subunit of GABA_A receptor. The primers of γ2 subunit were designed using online NCBI’s facility and cDNA sequences of the gene taken from
Gene Bank.

The sequence of primers used was:

γ2 forward: 5'- AGC CCG GAA GTC TCT GCC CAA -3', γ2 reverse: 5'- CCT CCC GTG TCT CCA GGC TCC -3', β-actin forward: 5'-CCC AGA GCA AGA GAG GCA TC-3', β-actin reverse: 5'-CTC AGG AGG AGC AAT GAT CT-3'.

Taq DNA polymerase (Roche, Germany) was used for DNA amplification and reactions were set up according to the manufacturer protocol. The PCR reactions were incubated for 5 min at 94°C, followed by 25 cycles of thermal cycling (45 sec at 94°C, 45 sec at 55°C and 45 sec at 72°C). The final cycle was followed by a 5 min extension step at 72°C. The reaction parameters were adjusted to obtain a condition with a linear relation between the number of PCR cycles and PCR products and with linear relation between the initial amount of cDNA template and PCR product. According to the results obtained from these tests, 25 cycles of amplification were used for analyzing all samples. Then, PCR products were subsequently analyzed on 1.5% agarose LMMP (Roche, Germany) gel and bands were quantified by densitometry using Lab Works analyzing software (UVP, UK). The possibility of the presence of contaminating genomic DNA was ruled out by using the yield of reverse transcriptase-minus (RT⁻) reaction, instead of cDNA template which caused no DNA amplification (data not shown). Finally, a semiquantitative PCR technique was used to estimate the levels of γ2 subunit of GABA_A receptor mRNA in tissue samples, normalized to an internal standard (β-actin).

Statistical analysis

The results are expressed as mean ± SEM. The differences in the mean of data between groups were determined by one-way ANOVA, followed by the Newman–Keuls test. P<0.05 was considered significant.

Results

The effect of allopregnanolone (ALLO) on serum glucose levels and body weight in diabetic animals

Diabetic rats developed hyperglycemia within one week after streptozotocin injection and serum glucose concentrations were significantly increased in diabetic animals compared with control animals (P<0.001). The levels of glucose remained at high levels 7 week after STZ injection. The data showed a significant change (increase) in the weight of control rats during the time curse of the study. At the end of the study (week 7), the weights of diabetic group were significantly lower than those at the start of study (Table 1). Treatment with ALLO (5 and 20 mg/kg/day for 7 weeks) had no attenuating effect on the serum glucose of the diabetic animals (Table 1). The weights of diabetic rats that received 5 and 20 mg/kg ALLO did not significantly decrease during the time course of the study and were higher than those in diabetic non-treated and vehicle-treated animals.

The effect of ALLO on nociceptive threshold in diabetic animals 

As shown in Figure 1A, diabetic animals showed a significant decrease in the nociceptive threshold (hyperalgesic response). The hyperalgesia appeared 2 weeks after STZ injection and persisted almost unchanged for up to the end of study (week 7). However, 5 and 20 mg/kg ALLO attenuated diabetes-induced hyperalgesia (Figure 1A).

At the end of the study, the tail flick latency was significantly (P<0.001) lower in diabetic rats (5.54±0.13) as compared to the control animals (2.69±0.19). However, administration of ALLO at doses of 5 and 20 mg/kg significantly prevented the induction of diabetes-induced thermal hyperalgesia in the 7^th week of the test (Figure 1B).  

The effect of ALLO on diabetes induced motor deficit

Rota-rod test has been conducted to assess motor coordination of rats (20). Diabetic rats showed significant reduction in their motor coordination and their ability to maintain themselves over the rota rod apparatus was significantly decreased (P<0.001) as compared to the control non-diabetic animals. Treatment of diabetic animals with 5 and 20 mg/kg of ALLO increased retention time to 85.5 and 76.3 % of the control value, respectively (Figure 2).

The effect of ALLO on γ2 mRNA in diabetic animals

Ethidium bromide staining of PCR products showed a single band of the expected size for β-actin (830 bp) and forγ2 subunit (337 bp). Diabetic animals showed a significant decrease in γ2 mRNA