Gender-Specific differences in pulsed magnetic field exposed to diabetic neuropathic rats

Diabetes Mellitus (DM) is an autoimmune disease, in which the circulating glucose levels increase due to either pancreatic β-cells cannot produce sufficient insulin or the cells of the body cannot effectively utilize insulin. There are usually two types of diabetes; Type 1 diabetes (insulin-dependent diabetes) is common in childhood or adolescence, while Type 2 diabetes (non-insulin-dependent diabetes) develops in the elderly with obesity or insulin resistance.
        
In Type-1 diabetes, as the pancreatic islet beta cells are damaged, the level of insulin produced by the beta cells decreases, accordingly the blood glucose levels increase. The kidneys filterout glucose together with the urine to reduce elevated blood glucose level which in turn increase the urine volume therefore leading intensive water loss in the body. When there is not sufficient insulin in the cellular environment, glucose cannot enter the cell. Thus, the cells have to find an alternative source of energy, which results in the activation of fatty acid metabolism causing an increase in the ketone levels, which also increase acidosis, leading ketoacidosis and if there is no insulin administered from other resources the consequences can be fatal (Aba and Asuzu, 2016, Kannan et al., 2016).
        
There are experimental and clinical studies reporting that magnetic field can cause positive progress  in various aspects of the body, including increased cell membrane permeability. Further, magnetic field has been shown to regulate hormonal and enzymatic processes of vasodilatation (regulation of blood flow by expanding blood vessels) and anti-inflammatory mechanisms as well as healing processes (with cell renewal effect) in a positive manner (Cevik et al., 2017, Khajuria et al., 2018). Diabetes mellitus is particularly giving rise to damage and dysfunction of many organs such as eye, kidney, nerves, heart and blood vessels. Magnetic field therapy has been employed non-invasively in the alternative treatment of diabetes with ameliorating effects on blood glucose level, weight loss ratio, and pain management, however research is still continuing on clarifying the mechanism of its actions.
        
Clinical researches are showing both male and female models of diabetes is required, as the varying responses of gender to drug so environmental factors is a likely consequence of pre-clinical studies primarily utilizing male mouse or rat models to avoid the distinct differences in female physiology and metabolism. Although female rat and mouse models of diabetes are readily available, reports on the gender specific differences observed in diabetic complications are limited, in the case of diabetic neuropathy. The characterization of diabetic neuropathy in gender-specific rat is important to establish the differences in physiological profiles between the sexes, including time of onset, degree of severity, and response to disease modifying agents. (O’Brienet_al2016).
        
Therefore, the purpose of our study was to evaluate the possible effects of PMF on weight loss ratio, blood glucose level and thermal and mechanical plantar tests used in the evaluation of diabetic neuropathy according to gender specific differences.

Experimental animals
 
In our study, 30 female Wistar Albino rats of 250-300 g weight were obtained from Cukurova University Experimental Medical Research and Application Center. The care of animals and all experimental procedures were in compliance with the experimental protocols of Cukurova University Animal Ethics Committee. The experimental groups consisted of 12 groups; C(F): Control female rats, C(M): Control male rats, DM(F): Diabetic female rats, DM (M): Diabetic male rats, C(F)-PMF; exposed to pulsed magnetic field control female rats, C(M)-PMF; exposed to pulsed magnetic field control male rats, DM(F)-PMF; exposed to pulsed magnetic field diabetic female rats, DM(M)-PMF; exposed to pulsed magnetic field diabetic male rats, DM(F)-INS; controlled-diabetic female rats, DM(M)-INS; controlled-diabetic male rats, DM(F)-INS-PMF; exposed to pulsed magnetic field controlled-diabetic female rats and DM(M)-INS-PMF; exposed to pulsed magnetic field controlled-diabetic male rats.The rats were kept at 23-24°C, with 12-hour light / darkness, 30-40% humidity in accordance with circadian rhythm. Rats were fed with water and a regular diet ad-libitum for six weeks.
 
Experimental diabetes mellitus in rats
 
Experimental diabetes was formed by intravenous injection of streptozotocin (STZ; 45 mg / kg) into the tail vein in rats. Approximately 48 hours after STZ injection, blood samples were withdrawn from the tail vein and blood glucose level of ≥ 300 mg/dl was considered as diabetic.
 
Controlled- Diabetic in rats
 
To reduce the complications risk in diabetes, glycemia was kept under controlled. In order to prevent sudden fall and rise in blood sugar levels and unwanted loss of body weight, long-acting insulin-glargine [2-3 U / 100 g (IG; Lantus®) A-ventisPharma, Germany, 24 hours] was administered at any time of day subcutaneously (Controlled-diabetic group [DM(F)-INS or DM(M)-INS]. Throughout the treatments the weights and blood glucose levels were measured regularly once a week at the same time of the day.
 
Pulsed electromagnetic field (PMF) therapy
 
The rats in the experimental pulsed magnetic field groups were exposed to PMF, which consisted of four consecutive pulses train (1, 10, 20, 40 Hz) at a strength of 1.5 mT 1h/5days/month in the horizontal direction of Helthmotz coil in three consecutive periodic pulses. In the pulsed magnetic field (PMF) application device, commonly used Helmholtz coils were used to create a uniform magnetic field. Helmholtz coils of 60 cm in diameter, 30 cm in length, were placed in a Faraday cage measuring 90x90x50 cm, then a 30x30x25 cm plexiglass box was placed in the middle region between the coils. The plexiglass cages were exposed to PMF 1 hours daily either in the morning and 09:00 to 11:00 or in the afternoon 13:00 to 15:00 (Fig 1).
 

 
Thermal plantar test
 
The thermal latency measurements were performed with thermal plantar test using Hargreave’s Method. Thermal latency measurements were made with the plantar test unit (MAY PWAM0903). The moving radiant heat source (8V-50W halogen bulb) probe under the glass layer was stimulated thermally (an infrared light source) from the center of the plantar region of the rat’s hind paw. When the rat sensed pain and pulled his foot, a photocell detected the interruption of reflected light, allowing  the infrared generator to turn off automatically and the time counter to stop there by determining the latency time (cut off time; 25 s).
 
Mechanical plantar test (Aesthesiometer)
 
Mechanical plantar tests are performed using Dynamic Aesthesiometer (Dynamic Plantar Aesthesiometer 37450 UGO BASILE-ITALY). When the rat pull its paw, the mechanical stimulation was automatically stopped and the force was recorded (Mechanical Threshold). These withdrawal responses were repeated at least 5 times with 10 seconds intervals (cut off time; 50 gr).
 
Statistical analysis
 
The results were expressed as ± standard error. Thermal latency and mechanical threshold values recorded before and after insulin treatment. PMF applications were evaluated by One-way ANOVA test. p<0.05 were considered statistically significant.

Comparison of weight percentages of female and male rats
 
The weight loss rate caused by diabetes at the end of six weeks decreased with PMF in both male and female diabetic rat which was statistically significant (p<0.05) (Fig 2).
 

        
Weight loss was not observed in PMF exposed control groups. In diabetics, PMF decreased weight loss caused by diabetes. In the controlled diabetic groups, the weight loss rate decreased with both insulin and PMF effects. The results were statistically significant.
        
In DM groups exposed to PMF for six weeks, the reduction in weight loss rate suggests that water retention is possibly related osmotic diuresis and diabetic hyperglycaemia (Macias et al., 2000; Tasset et al., 2012).
 
Comparison of glucose percentages of female and male rats
 
At the end of six weeks, high blood glucose, which was caused by diabetes, decreased in both female and male diabetic rats with PMF treatment (Fig 3). Reduction in blood glucose level in female or male diabetic rats were statistically significant (p<0.05). Blood glucose levels under the influence of PMF decreased in DM(F) or DM(M) groups with PMF. Decreased blood glucose levels in female diabetic groups exposed to PMF was more pronounced in male diabetic groups possibly due to different hormones and metabolism of females. Ocal et al., (2008) found that low-frequency PMF partially reduced the elevated blood glucose level due to diabetes. It has also been shown that low frequency PMF causes anti-hyperglycemic activity in diabetic rats, but PMF administration did not significantly alter hyperglycemia. Our findings are also compatible with the results of Lei et al., (2013). The different effects of low frequency PMF on hyperglycemia in experimental diabetic rats may be due to different frequency, intensity, duration and direction of PMF (Basset, 1993; Sisken et al., 1993; Walker et al., 1994). In diabetic rats, the anti-hyperglycemic effect of low-frequency PMF may be caused due to alterations in cell membrane and tissue glucose uptake owing to PMF (Sima and Kamiya, 2006; Laiti-Kobierska et al., 2002; Tartakoff, 1980; Gorczynska and Wegrzynowicz, 1991; Bellossi et al.,1998). PMF can also affect biological systems physiologically, structurally or functionally. In diabetic conditions, various structural and functional abnormalities can occur at the molecular level including signal mechanisms and chemical processes of cells. PMF applications can optimize the system and lead the therapeutic effects by reorganizing the cells thereby restoring the cellular functions (Basset et al., 1989).
 

 
Comparison of mechanical threshold values in female and male rats
 
The mechanical threshold value decreased in both male and female groups in diabetic groups, but increased after six-week of PMF exposure which is statistically significant. The positive effect of PMF was observed more clearly in controlled-diabetic groups by standard insulin treatment (DM (F) -INS or DM (M) -INS). Which was statistically significant in diabetic neuropathic rats (Fig 4) (p<0.05).
 

 
Sensitivity to painful and painless stimuli changes because of sensory dysfunction in diabetic neuropathy (Canedo-Dorantes et al., 2015; Blank et al., 2009). Diabetes impair the sensory nerve structures and functions depending on the level of hyperglycemia along with abnormal sense indications such as mechanical allodynia and thermal hyperalgesia (Marcias et al., 2000, Schmader 2002).
 
Comparison of thermal latency in female and male rats
 
Increased thermal latency values with diabetic neuropathy decreased with PMF treatment after six weeks. which was were statistically significant (Fig 5). The decrease in thermal latency with PMF was statistically significant in male (p<0.05), but not in female diabetic neuropathic rats.
 

        
Mert et al., 2010, found that in streptozotocin induced acute and chronic diabetic rats, PMF caused significant alterations in thermal latency and mechanical threshold values, indicating that PMF may have a therapeutic efficacy. Lei et al., (2013) have reported that 15 Hz PMF alleviates peripheral neuropathic symptoms in diabetic rats and significantly inhibits the development of hypersensitivity to mechanical and painful thermal stimuli (Lei T. et al., 2013). In other scientific investigations of PMF applications in painful diabetic peripheral neuropathy (DPN) animal models, it has been observed that PMF may prevent and possibly reverse the development of sensory abnormalities triggered by diabetes (Pieper et al., 2010; Goudarzi et al., 2010; Navratil and Hlavaty, 1993; Li et al., 2013; Quittan et al., 2000, Schmader 2002).

The sequential low frequency magnetic field does not have a significant effect on healthy rats but has been shown to  have a therapeutic effect in diabetes-induced rats which could be used as an alternative therapy that can be used in the treatment of diabetic neuropathic pathologies of PMF (Quittan et al., 2000; Pieper et al., 2010). This non-invasive method does not cause any side effects as pharmacological drugs do and contributes to the elimination of vascular effects of diabetes. However, the gender should always be taken into account when evaluating the efficacy of this method. In our settings, metabolism and physiological differences between male and female rats affected our results. In particular, the influence of DM, on hormonal levels can be explained by the fact that female are the reason for preference in experimental studies. For this reason, the frequency, intensity and direction of the applied PMF should be adjusted according to gender. In conclusion, PMF’s gender specific actions particularly on insulin release and recruitment mechanisms together with its effects on hormonal pathways, stress factors and free radicals should be investigated with further studies.