Research Design and Methods
Male Sprague-Dawley rats that weighed 250 ± 5 g at the beginning of the study were used. Experimental diabetes was produced by the intraperitoneal injection of streptozotocin (STZ) in 0.05 mol/l citrate buffer at pH 4.5 (65 mg/ml; dose 1.32 ml/kg). The control group received an intraperitoneal injection of citrate buffer alone. Control and diabetic rats had free access to Purina Rodent Laboratory Chow and water. They did not receive insulin treatment. The rats were accepted as diabetic when their fasting blood glucose exceeded 16.7 mmol/l 3 days after injection of STZ and remained >16.7 mmol/l at the time when they were killed. Several groups of rats were used to accomplish these studies.
Duration of diabetes was different for three groups:
EDN, duration 1 month (n = 8); age- and gender-matched control littermates (n = 8)
EDN, 3 months (n = 8); age- and gender-matched control littermates (n = 8)
EDN, 12 months (n = 9); age- and gender-matched control littermates (n = 9)
EDN, 12 months (n = 5); age- and gender-matched control littermates (n = 5).
Nerve conduction studies were done on groups 1-4 and one EDN and one control from group 4. Groups 1-3 were used for immunohistochemical studies (8-hydroxy-2'-deoxyguanosine [8-OHdG] and caspase-3) and for TUNEL. Group 4 was used for histochemical studies of cytochrome oxidase reactivity.
We used methods that are standard in our laboratory. Nerve was fixed in situ for 30 min, flash frozen, and cut into 10-µm sections. Endogenous peroxidase, avidin, and biotin were blocked appropriately, and a 0.05% Tween-20 in phosphate buffer was used. Sections were incubated in the primary antibody for 1 h. After washing, specific immunoreactivity was visualized using a Vectastain Elite ABC kit (Vector Labs, Burlingame, CA) with diaminobenzidine as the chromagen. Negative controls were generated by omission of the primary antibodies and the use of a blocking peptide. Specificity for 8-OHdG was additionally tested by a pretreatment with an RNase solution that did not reduce staining and a DNase-1 (Boehringer Mannheim) treatment that reduced staining to background staining intensity. We used the following antibodies: anti-8-OHdG (mouse monoclonal, 1:40; Genox, Baltimore, MD) and anti-caspase-3 (rabbit polyclonal, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA). Staining for cytochrome oxidase was performed using methods from Seligman et al.[12]. A TUNEL labeling kit (Oncogene, Boston, MA) was used for labeling apoptotic nuclei. Positive and negative controls were generated per kit protocol. An additional positive control was also used by use of DNase-1 to "nick" the DNA. Three levels of DRG were assayed per animal at 100-µm intervals. An average of 100 neurons/1 month, 104 neurons/3 months, and 125 neurons/12 months were graded per animal.
For 8-OHdG and caspase-3, we used a semiquantitative grading system, because cells varied by the severity and presence of staining. To a first approximation, the grades were as follows: 0, light staining and only affecting ≤5% of neurons; 1, staining affecting 5-10% of neurons; 2, staining affecting 10-20% of neurons; 3, staining affecting ≥20% of neurons. In the evaluation of TUNEL positivity, neurons were considered positive when the nuclei stained positively and showed chromatin clumping. The number of neurons that fulfilled the criteria was expressed as a percentage.
Electrophysiology. We used techniques that are standard for our laboratory[13,14]. Sensory nerve conduction velocity of digital and caudal nerves was measured using fine stainless steel near-nerve-stimulating and -recording electrodes. Motor nerve conduction velocity was measured in the sciatic-tibial and caudal nerves. The compound muscle action potentials were recorded from the pair of fine stainless steel electrodes in the dorsum of hind paw while stimulated by another pair of electrodes at the level of the sciatic notch and ankle. Recordings were made at 35°C, amplified 1,000 times, stored on computer disks, and analyzed off-line using a Nicolet 4094 digital oscilloscope (Nicolet Instruments, Madison, WI) with associated stimulators and stimulus isolation units.
Means of continuous variables were compared between groups using ungrouped two-tailed t test. Nonparametric relationships were examined with Mann-Whitney U tests. Univariate associations between variables were performed using Spearman rank correlations. Data were expressed as mean ± SE unless otherwise stated. P < 0.05 was considered significant.
Diabetes. 2003;52(1) © 2003 American Diabetes Association, Inc.
Cite this: Oxidative Injury and Apoptosis of Dorsal Root Ganglion Neurons in Chronic Experimental Diabetic Neuropathy - Medscape - Jan 01, 2003.
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