The experimental animals were male Wistar rats (200-300 g body weight) purchased from the animal house of Iran University of Medical Sciences. They were kept under standard conditions (Temperature 22±2℃, the relative humidity of 60±5%) and 12 h light/dark cycle. They had free access to a standard pellet diet and water ad libitum. The experiments were approved by the ethical committee of Iran University of Medical Sciences. (96-01-118-29912).
Diabetic neuropathy in type 1 diabetic streptozotocin model
Diabetes mellitus (DM) type 1 was induced through a single intra-peritoneal injection of dissolved streptozotocin (Sigma, USA) (55 mg/kg) in normal saline following 12 h fasting. After 72h of injection, the fasting blood sugar (FBS) levels were measured from animals’ tails using a glucometer. The FBS over 250 mg/dl was considered as DM and 5 weeks following confirmed hyperglycemia, DN occurred (Davidson et al. 2011).
After confirmation of hyperglycemia, the rats were divided randomly into 5 groups, each group consists of 7 animals (n=7): (1) Control group which received DMSO (drugs solvent), orally, 5 days/week for 5 weeks; (2) DN group: diabetic rats which received 0.5% DMSO, orally, 5 days/week for 5 weeks; (3) DN + rolipram group: diabetic rats which received rolipram (1mg/kg), orally, 5 days/week for 5 weeks; (4) DN+ pentoxifylline group: diabetic rats which received pentoxifylline (100 mg/kg), orally, 5 days/week for 5 weeks ; (5) DN + rolipram +pentoxifylline group: diabetic rats which received a combination of rolipram (0.5mg/kg) and pentoxifylline (50 mg/kg), orally, 5 days/week for 5 weeks. All oral treatments were performed by gavage. We selected doses of rolipram and pentoxifylline according to the literature. Among the effective doses of rolipram in previous studies (1-3 mg/kg), due to the side effects of this drug, lower dose (1 mg / kg) was selected in this study. (Mokry et al.2013; Han et al. 2012; Kajana et al. 2008). According to previous studies, the appropriate dose of pentoxifylline 100 mg / kg was chosen. (Neves et al. 2015; Halis et al. 2019). In previous studies, the dose of rolipram in combination with another phosphodiesterase inhibitor was halved. Therefore, the first we chose rolipram dose (0.5 mg/kg) and then adjusted pentoxifylline similar dose (50 mg/kg) (Mokry et al, 2013).
Dorsal root ganglion (DRG) isolation
After the end of treatment period, animals were anesthetized using ketamine hydrochloride 10% (60 mg/kg) and xylazine hydrochloride 2% (8 mg/kg), then the spines were broken and the whole vertebrates were separated, DRG neurons isolated from the second cervical (C2) to second lumbar (L2) spine region on both sides. DRG neurons was fixed by 10% formalin for histological study and the others immediately transferred to liquid nitrogen for further evaluation (Hosseini et al. 2011).
Processing of DRG neurons for further analysis
DRG neurons were placed in lysis buffer (10mM Tris-base, 150mM NaCl, 1mM EDTA, 1% NP40 in double distill H2O) including suitable enzyme inhibitors on ice. Then the tissues were homogenized using a mechanical grinding pestle for 30 s on ice after that were centrifuged for
15 min at 16,000×g at 4 °C and finally the supernatant was collected as sample for further analysis (Galeshkalami et al. 2019).
cAMP level was measured using cyclic AMP ELISA kit (Cayman chemical) according the manufactures’ instruction. The level of cAMP in samples were assessed at 405 nm by a plate reader (Synergy HT, BIOTEK, USA).
Measurement of reactive oxygen species (ROS)
For assessment of ROS generation, samples were incubated with a fluorescent dye 2', 7-dichlorofluorescein diacetate (DCFH-DA) at 37℃ for 30 min. The intensity of fluorescence was measured at 485 and 528 nm as excitation and emission wavelengths respectively, by a Microplate Reader (BIOTEK instruments, USA) (LeBel et al. 1992).
Measurement of lipid peroxidation (LPO)
The Lipid peroxidation was measured using the thiobarbituric acid reactive substances (TBARS) method. The samples were mixed with thiobarbituric acid (TBA) and then were heated in boiling water for 30 min. The reaction of (TBA) and MDA produced red color which its absorbance was measured at 532 nm using a Microplate Reader (BIOTEK instruments, USA) (Ohkawa et al. 1979).
Measurement of total antioxidant capacity (TAC)
TAC was determined using the FRAP method. The basis of FRAP method is a reduction of Fe3+ to Fe2+ by the samples. The complex of Fe2+ and 2,4,6-tris (2-pyridyl)-1,3,5-triazine (TPTZ) generates a blue color which its absorbance was taken at 593n nm through spectrophotometer (Benzi et al. 1999).
Measurement of superoxide dismutase (SOD) activity
The activity of CAT was measured through assay kits (Cayman Chemical, Ann Arbor, USA). The o.-2 which is generated from xanthinoxidase activity reacts with 2-(4-iodophenyl)-3-(4-nitrophenol) 5-phenyl tetrazolium chloride (INT) and produced a red formazan dye. The SOD activity was determined through reaction inhibition from one unit of SOD activity was defined as the amount that leads to 50% inhibition of the rate of reduction of INT. The color reaction was measured at 505 nm by ELISA microplate reader. The results are presented as unit per milligram of protein.
Measurement of catalase (CAT) activity
The CAT enzyme activity by observing the disappearance of hydrogen peroxide in the spectrophotometer at 240 nm was determined. Briefly, supernatant (100 μL) and alcohol ethanol (10 μL) were vortexed and placed in ice water bath for 10min. Then, 10 μL of Triton X-100 was added to mixture and was vortexed in room temperature. Thereupon, 100 μL of phosphate buffer (H2O2 0.66 M) was added to the mixture, and decrease of absorbance was measured at 240 nm by spectrophotometer (Cecil CE7250-7000 series, Milton Technical Centre, Cambridge, UK). The results were presented as μ/milligram protein (Sinha et al. 1972).
Measurement of total thiol groups
The procedure of determination of total sulfhydryl was described previously (Hu et al. 1994). 0.2 ml from sample was mixed with 0.6 ml of Tris-EDTA buffer [Tris base (0.25 M), EDTA (20 Mm), PH 8.2] in a 10 ml test tube, after that mixed with 40 ml of DTNB (10 mM) in methanol. 16 ml of methanol added until the final volume reaches 4 ml. After capping, the test tube was centrifuged at 3000 g for 10 min and after 15-20 min the color appeared and the supernatant absorbance was measured at 412 nm.
Measurement of cyclooxygenase 2 (COX-2)
COX-2 was detected using rat cyclooxygenase-2, ELISA Kit, Cusabio (Houston, TX, USA) according the manufactures’ instruction.
Measurement of Tumor necrosis factor-α (TNF-α)
For assessment of TNF-α level, a Tumor necrosis factor-α kit (Zell bio Gmbh, Germany) was used. TNF-α level in samples were assessed at 450 nm using a plate reader (Synergy HT, BIOTEK, USA).
DRG neurons were subjected to quantitative RT-PCR experiments. Total RNA was isolated from DRG tissues by using Trizol reagent in accordance with the manufacturer’s protocol. The extracted RNA (1 μg) was reverse-transcribed into cDNA by using a PrimeScript RT reagent kit. RT-PCR was performed. The primer sequences of NF-kB: Forward primer: TTCAACATGGCAGACGACGA, Reverse primer: AGGTATGGGCCATCTGTTGA.
After PCR was completed, the products were analyzed through electrophoresis on 1.2% agarose gel and photographed under UV light in an EC3 Imaging System (UVP, Upland, CA) (Miroliaee et al. 2011).
Measurement of motor function
Motor function was assessed by open-field activity tests. The rats were placed in an open-field box where velocity (cm/sec.) and distance moved (cm) were recorded with a camera on the top of the box for 5 min. The EthoVision tracking system (Noldus Information Technology, Wageningen, the Netherlands) was used to measure motor function by determining speed and distance of animal movement (Hosseini et al. 2011).
Isolated DRG neurons were fixed in the paraformaldehyde and then embedded in paraffin and after that were cut into 40 μm sections. These sections were stained with hematoxylin and eosin (H&E) and were used for microscopic analysis by Olympus microscope (LX71, Japan).
The results were presented as the mean ± SEM and were analyzed using one-way ANOVA and Tukey's post hoc tests. p < 0.05 was considered as statistically significant.