Molsidomine ameliorates diabetic peripheral neuropathy complications in Wistar rats

Abstract Diabetic neuropathy is a disorder that affects various regions of the nervous system and there is no specific treatment available for it. This study evaluated the protective effect of molsidomine in diabetic neuropathy in rats. Diabetes was induced in male Wistar rats by administrating streptozotocin (52 mg/kg ip). Diabetic rats were treated with molsidomine 5 mg/kg po and 10 mg/kg po. After 8 weeks of treatment, motor coordination, mechanical allodynia, mechanical hyperalgesia, nerve conduction velocity, and glycosylated hemoglobin were assessed. Thereafter, animals were killed and the sciatic nerve was isolated for measurement of reduced glutathione and lipid peroxidation, and histopathological analysis. Treatment with molsidomine significantly improved motor coordination, paw withdrawal threshold, mechanical threshold, and nerve conduction velocity. Furthermore, molsidomine treatment also reduced malondialdehyde levels and prevented depletion of reduced glutathione in the sciatic nerve homogenate. Histopathology revealed that molsidomine treatment maintained normal architecture of the sciatic nerve. The results of our study strengthen the alternative use of molsidomine in diabetic neuropathy.


| INTRODUC TI ON
Diabetes mellitus (DM) prevalence is increasing at an alarming rate as it was 381 million people globally in 2013 and is estimated to be 463 million people in 2019 rising to 578 million by 2030. It is a disease with high rate of complications such as neuropathy, nephropathy, retinopathy, erectile dysfunction etc. 1,2 Diabetic neuropathy is a family of disorders that damage the different regions of the nervous system, either individually or in combination. It affects pain fibres, motor neurons and autonomic nervous system. 3 It results in large economic costs for its care. [4][5][6] The various kind of neuropathies include peripheral neuropathy, proximal neuropathy, autonomic neuropathy and focal neuropathy. 7 There are a number of reasons for the pathogenesis of diabetic neuropathy and polyol pathway of glucose metabolism is thought as one of the major mechanism. 8 Peripheral neuropathy is a type of nerve damage that usually affects feet and legs and sometimes hands and arms. 9 It is proved that reactive oxygen species (ROS) plays a significant role in the pathophysiology of neuropathic pain in diabetes. 10 Out of all diabetic patients, 50% of patients develop neuropathy and painful neuropathy ranges from 10% to 20% in patients with diabetes. Diabetic patients can experience nerve problems at any time and the problem increases with age, weight and duration. 5 The complications across various countries varies from 10% to 30% and it is higher in developed countries than in developing countries. These complications can lead to painful symptoms and can affect quality of life of the patient. The treatment for the painful diabetic neuropathy is mainly focused on pain control. The treatments include a number of antidepressants, anticonvulsants, topical agents such as capsaicin cream, lidocaine patches and isosorbide dinitrate topical spray have also been tried but the benefits were uncertain. Since there is lack of exact medication for neuropathy, research is still carried out. 2 Nitric oxide is an endogenous vasodilator which act as a neurotransmitter which is produced from L-arginine using an enzyme nitric oxide synthase. In diabetic patients, hyperglycemia stimulates the over production of ROS such as superoxide anion which reacts rapidly with nitric oxide radicals forming peroxynitrite anion, which is a toxic oxidant capable of damaging neurons leading to neuronal injury. 11,12 Molsidomine is a vasodilating and antianginal agent. Since it is a prodrug, it is converted into its metabolite 3-morpholinosydnonimine (SIN-1), which spontaneously provides nitric oxide. Nitric oxide has a significant impact on tissue injury, inflammation, vasodilation, cell defence as well as for regulating cerebrovascular hemodynamics. Nitric oxide also has following characters like antioxidant, antiapoptotic and anti-inflammatory activity. It can be used to treat the eye tissue damage caused due to ionising radiation. [13][14][15] Till now, there is no data of molsidomine acting against diabetic peripheral neuropathy. So, the present study was designed to evaluate the protective effect of molsidomine against diabetic peripheral neuropathy.

| Animals
Wistar rats of appropriate age of either sex weighing about 250-300g were used for the study. The animals were housed in large propylene cages in an air-conditioned room at 24 ± 1°C with a 12 hr light/dark cycle and allowed ad libitum access to water and standard diet. Paddy husk was used as bedding material. The use of animals for the experiments were approved by Institutional Animal Ethics Committee (IAEC, reference number: AACP/IAEC/Dec2016/05) and Committee for the Purpose of Control and Supervision of Experimental Animals guidelines were followed.

| Induction of diabetes
STZ was administered at a dose of 52mg/kg body weight intraperitoneally to induce diabetes. Glucose solution was given a day after STZ injection. After 72 hours of STZ injection, blood samples (from tail) were collected after overnight fasting. Animals with fasting blood sugar >250mg/dL was considered as diabetic and divided into the following groups.
Non diabetic rats were assigned as the normal control group

| Motor coordination
Motor coordination was evaluated by Rota-Rod tread mill. Rats were initially trained to remain themselves on the rotating rod for more than 2 minutes. In the test session the rats were placed in the rotating rod and the latency to fall was recorded. 16

| Mechanical allodynia
Mechanical allodynia was performed as per the method of Yamamoto H et al using Von Frey apparatus. 17

| Mechanical hyperalgesia
The measurement of mechanical nociceptive threshold was measured using fabricated Randall Selitto paw pressure device which applies a linearly increasing mechanical forcing to the dorsum of the rat's hind paw. 18

| Nerve conduction velocity
Non-invasive nerve conduction velocity (NCV) was measured using power lab data acquisition system. The rats were anaesthetized with ketamine:xylazine(80-100 mg/kg :5-10 mg/kg i.p.) and during the experiment the body temperature of animal was maintained.
The sciatic nerve was stimulated with supramaximal stimuli (8V) at 20 Hz. The latencies of the compound muscle action potentials were recorded via bipolar electrodes from the first interosseous muscle of the hind paw and measured from the stimulus artifact to the onset of the negative M-wave deflection. Motor NCV was calculated by subtracting the distal latency from the proximal latency and the result were divided by the distance between the stimulating and recording electrode.

| Estimation of glycosylated hemoglobin
Blood was withdrawn from retro orbital of rat and collected in EDTA tubes. The glycosylated hemoglobin (GHb) was determined by using commercially available kits. 19

| Sciatic nerve homogenate preparation
The rats were sacrificed by overdose of anaesthesia. A segment of sciatic nerves was isolated. Sciatic nerve samples were rinsed with ice cold saline (0.9%w/v sodium chloride) and homogenised in chilled phosphate buffer (pH 7.4). The homogenate thus obtained was used for measurement of reduced glutathione and lipid peroxidation.

| Estimation of reduced glutathione
The sciatic nerve was dissected out and washed with saline, chopped over ice and homogenates were prepared with 0.1 mol/L phosphate buffer. Glutathione was quantified by the method of Moron et al. 20

| Histopathology
Sciatic nerve was used for histopathology to observe the changes in the cell architecture using H&E (Hematoxylin and Eosin) stain.

| Statistical analysis
All data were expressed as mean ± SEM and analysed with one-way analysis of variance between the groups and followed by Tukey's Multiple Comparison Test were used to assess differences between the groups. Probability values *P < .05, **P < .01, ***P < .001 were considered significant.

| Effect of molsidomine on motor coordination
The diabetic rats showed a significant decrease in the motor coordination as compared to normal rats (P < .001). Pre-treatment with molsidomine improved the retention time as compared to diabetic rats. (Figure 1).

| Effect of molsidomine on mechanical allodynia
The diabetic rats showed a significant reduction in paw withdrawal threshold as compared to normal rats (P < .001). Pre-treatment with molsidomine improved paw withdrawal threshold as compared to diabetic rats (Figure 2).

| Effect of molsidomine on mechanical hyperalgesia
Diabetic rats showed significant reduction in mechanical threshold as compared to diabetic rats (P < .001). Pre-treatment with molsidomine improved mechanical threshold as compared to diabetic rats ( Figure 2).

| Effect of molsidomine on sciatic nerve conduction velocity
The nerve conduction velocity was significantly reduced in diabetic control rats when compared to normal rats (P < .001). Pre-treatment with molsidomine shows significantly improved in NCV when compared with diabetic rats (Figure 3).

| Effect of molsidomine on glycosylated hemoglobin, reduced glutathione and lipid peroxidation
Diabetic rats showed a significant increase in percentage of GHb and MDA levels as compared to normal rats (P < .001). Pre-treatment with molsidomine significantly reduced percentage of GHb and MDA levels as compared to the diabetic control rats (P < .001).
Whereas diabetic rats showed a significant reduction in sciatic nerve reduced glutathione content as compared to normal rats (P < .001).

Fall of time(Sec)
Pre-treatment with molsidomine significantly improved glutathione levels as compared to diabetic rats (P < .001) ( Table 1).

| Histopathology
Sciatic nerve of normal rats stained with hematoxylin-Eosin(40X) shown longitudinal section showing the elongated Schwann cell nuclei and longitudinally oriented axons with myelin sheath ( Figure 4A).

Sciatic nerve of diabetic rats stained with hematoxylin-Eosin(40X) has
shown mainly axonal swelling observed with intact myelin ( Figure 4B).
Sciatic nerve of rats treated with molsidomine 5 mg/kg showed nuclear degeneration in focal areas with few fibre arrangements ( Figure 4C) whereas rats treated with molsidomine 10 mg/kg showed nuclear degeneration in few areas with few fibre arrangements ( Figure 4D).

| D ISCUSS I ON
Diabetic neuropathy has to be identified in early stages to prevent secondary complications such as neuropathic pain and diabetic foot. 22 The most commonly used animal for painful diabetic neuropathy is STZ induced diabetic rats. Mechanical allodynia and mechanical hyperalgesia are the common endpoints used to assess analgesic activity of drug in animal model. In our study mechanical allodynia and mechanical hyperalgesia were employed to assess the withdrawal threshold of the rat hind paw. The withdrawal threshold was improved following the treatment with molsidomine showing the analgesic activity of the drug. 17 In STZ induced diabetic rats causing hyperalgesia is often ac- radical. This superoxide anion radical rapids reacts to form peroxynitrite.
Literature shows that treatment with molsidomine increases peroxynitrite concentration and decreases superoxide anion radical. 29 Histopathological analysis of sciatic nerves of diabetic rat showed mainly axonal swelling observed with intact myelin. Treatment with molsidomine significantly improved fibre arrangements and is dose dependent.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest.