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. 2022 Sep 28;5(10):985–992. doi: 10.1021/acsptsci.2c00140

Controversial Role of Folic Acid on Diabetic Auditory Neuropathy

Aida Doostkam †, Hossein Mirkhani ‡,*, Kamyar Iravani §,*, Saied Karbalay-Doust ∥, Afsaneh Doosti ⊥, Elham Nadimi #, Fatema Pirsalami ∇
PMCID: PMC9578138  PMID: 36268113

Abstract

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Objective: Diabetic auditory neuropathy (DAN) is a common complication of diabetes that seriously affects the quality of life in patients. In this study, we investigate the role of folic acid in the treatment of DAN in an experimental rat model. Methods: Thirty-two Sprague–Dawley rats were equally divided into four groups: group 1, normal; group 2, diabetic rats; and groups 3 and 4, diabetic rats treated with folic acid (40 and 80 mg/kg, respectively). We used some tools to investigate the therapeutic effect of folic acid on DAN. We evaluated auditory brain stem response (ABR), estimated the volume and number of spiral ganglion and the volume of stria vascularis and spiral ligament by the stereological method, and measured the blood levels of homocysteine (HCY), malondialdehyde (MDA), and superoxide dismutase (SOD). Results: Our study showed that folic acid treatment was not significantly effective in improving structural and functional disorders in DAN, even though its effectiveness in reducing HCY (P < 0.001) and MDA (P < 0.05) as oxidative biomarkers was significant. Conclusion: Folic acid is not effective in relieving morphological and functional disorders in DAN.

Keywords: auditory brainstem response, auditory neuropathy, diabetes mellitus, folic acid, histology

Diabetic neuropathy is a common complication of diabetes mellitus (DM), which affects the sensory, motor, and autonomic nerves and the central nervous system.1−3 Ischemia caused by damage to the feeding vessels of the nerves in diabetic patients causes neural fiber demyelination, degeneration, and the resulting neuropathy.2,4

The major metabolic disorders that cause vascular damage in diabetic patients, followed by neuropathy, are mainly due to the polyol pathway and oxidative stress, which increase free radicals, especially reactive oxygen species (ROS) in the tissues.4−6

Impaired neural microcirculation in the auditory system can cause morphological and functional disorders. Diabetic auditory neuropathy (DAN) is mainly accompanied by debilitating symptoms such as hearing loss, tinnitus, and dizziness.7,8

Due to the significant occurrence of neuropathy and its auditory type in diabetic patients, various agents have been evaluated to prevent and treat neural damage in experimental and clinical studies. Among the drugs that have been investigated for diabetic neuropathy include rutin,9 folic acid,10,11 alpha-lipoic acid,12 and benfotiamine (a vitamin B1 derivative).13 These investigations are not sufficient for clinically acceptable applications and results.

In our study, we applied rutin for the prevention and treatment of DAN in rats; the beneficial effect of this drug in terms of morphological and functional improvement was defined to some extent.9

One of the drugs under investigation, which is relatively more clinically used than others in diabetic neuropathy, is folic acid. Due to the discrepancies in animal and clinical studies and clinical observations that, in some cases, show the ineffectiveness of this drug, we set out to investigate folic acid effectiveness against DAN in rats, following our previous study on rutin.

In this study, auditory brain stem response (ABR), stereology of spiral ganglion, homocysteine (HCY), superoxide dismutase (SOD), and malondialdehyde (MDA) levels were used as defining criteria to investigate the effect of folic acid treatment in DAN.

Folic acid (vitamin B9) is a water-soluble vitamin synthesizing genetic materials, producing red blood cells, and promoting fetal health. Folic acid is a synthetic form of folate better absorbed than folate. The main sources of folate include leafy vegetables, citrus fruits, mushrooms, grains, and liver.14,15

As a donor of single carbon units, folate synthesizes serine from glycine, nucleotides from purine precursors, and deoxythymidylate monophosphate. In addition to its role in synthesizing DNA and messenger RNA, it acts as a methyl donor to produce methylcobalamin, which is used in the remethylation of HCY to methionine.16,17

Folic acid seems to have the potential ability to grow and differentiate neural cells. It has a neuroprotective property due to its effects on HCY metabolism, decreasing MDA levels and increasing the expression of nerve growth factors.18,19

ABR is one of the best tools for evaluating auditory neuropathy functionally. This method measures the evoked electrical activities in the auditory nerve and its brainstem connections. DM can cause changes in ABR potential latency and waveforms by disrupting the microcirculation of the auditory pathway.7,20

Impaired microcirculation in DAN and subsequent cochlear ischemia can cause morphological changes in the hair cells, spiral ganglion, spiral ligament, and stria vascularis.7 In this study, we used the stereology method in the spiral ganglion, spiral ligament, and stria vascularis to examine morphological changes in DAN and the response to treatment by measuring the volume and number of cells.

HCY is a non-proteinogenic amino acid made from the amino acid methionine following a transmethylation reaction. Many studies have shown an association between high levels of HCY in diabetic patients and its vascular and neurological complications.21−23

SOD is an antioxidant enzyme that catalyzes the dismutation of superoxide radicals into oxygen and hydrogen peroxide. This enzyme acts as a biomarker of antioxidant activity in controlling oxidative stress.24

MDA is a product of polyunsaturated fatty acids peroxidation. MDA is considered a reliable biomarker in oxidative stress measurement levels in many experimental and clinical studies.25,26

This study used ABR to evaluate the hearing function, the stereological method for morphological evaluation in the spiral ganglion, stria vascularis, and spiral ligament. Also, we applied oxidative biomarkers of HCY, MDA, and SOD levels as tools to evaluate the efficacy of folic acid treatment on DAN.

Results

Outcome of Folic Acid on the Function of ABR

After eight weeks of DM induction, the effect of diabetes on ABR wave forms was observed. The negative changes were significant in the latency of wave II, hearing threshold, and the presence of wave V (P < 0.001). The latency of wave II increased the hearing threshold, and the rate of presence of wave V is shown in Figure 1 and Table 1. Folic acid (40 and 80 mg/kg) had no significant recovery effect on ABR recordings at the end of the study (about eight weeks after the induction of diabetes) (Figure 1a,b and Table 1).

Figure 1.

Figure 1

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Latency of wave II (a) and hearing threshold (b) in the normal, DM, DM + FA40, and DM + FA80 groups. *** indicates P < 0.001 vs diabetic group.

Table 1. Types and Frequency of Wave V in the Normal, Diabetic, DM + FA40, and DM + FA80 Groupsa.

group wave V
 p-value
normal (%) with delay (%) absent (%)
normal (n = 8) 100 0 0 <0.0001
diabetic (n = 8) 0 0 100  
DM + FA40 (n = 8) 12.5 0 87.5 0.99
DM + FA80 (n = 8) 0 37.5 62.5 0.99
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a

Differences were tested by the Kruskal–Wallis and Dunn’s post hoc test. All data is in comparison to diabetic group.

Stereological Studies

Evaluation of Spiral Ganglion, Spiral Ligament, Stria Vascularis Volume, and Spiral Ganglion Neuron Count

There is a significant decline in the total volume of the spiral ganglion, spiral ligament, stria vascularis, and the number of spiral ganglion neurons in the rats with DM compared to the normal animals (P < 0.05) (Figure 2a,c,d,b).

Figure 2.

Figure 2

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The aligned dot plot of the volumes of the spiral ganglion (a), spiral ligament (c), stria vascularis (d), and the number of spiral ganglion neurons (b) in the normal, DM, DM + FA40, and DM + FA80 groups. Each dot represents an animal, and the horizontal bar is the mean of the listed parameters. * indicates P < 0.05 vs diabetic group.

The total volume of spiral ganglion, spiral ligament, stria vascularis, and the number of spiral ganglion neurons did not change significantly in DM + FA40 and DM + FA80 groups (Figure 2a,c,d,b).

Biochemical Assays

The erythrocyte level of SOD in the diabetic group was significantly lower than that in the normal group (P < 0.001). On the other hand, these values for folic acid groups (DM + FA40 and DM + FA80) were not higher than those of the diabetic group (Figure 3a).

Figure 3.

Figure 3

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Serum level of SOD (a), MDA (b), and HCY (c) in the normal, DM, DM + FA40, and DM + FA80 groups. * indicates P < 0.05 vs diabetic group. ** indicates P < 0.01 vs diabetic group. *** indicates P < 0.001 vs diabetic group. **** indicates P < 0.0001 vs diabetic group.

The MDA serum level in the diabetic group was significantly higher than that in the normal group (P < 0.01), but serum levels of MDA for folic acid-treated groups (40 and 80 mg/kg/day) were significantly lower than that of the diabetic group (P < 0.05) after the intervention (Figure 3b).

The serum level of HCY was increased significantly in the diabetic group compared to that in the normal group (P < 0.001; Figure 3c). Administration of folic acid 40 and 80 mg/kg/day showed a significant decrease in the serum level of HCY (P < 0.001 and P < 0.0001, respectively; Figure 3c).

Discussion

This study revealed that folic acid does not significantly affect the prevention and treatment of functional and structural deficits in DAN despite reduced MDA and HCY levels.

In this study, folic acid was administered at 40 and 80 mg/kg. Still, neither showed a significant response in ABR recordings and spiral ganglion stereology.

In our opinion, spiral ganglion stereology and ABR recordings are indicators that are more closely related to a clinical situation than MDA and HCY levels.

The advantage of our study is the histological examination through the stereological method and its compliance with functional outcomes in diabetic neuropathies treated with folic acid.

ABR is an essential tool for assessing hearing function. Numerous clinical and experimental studies have shown the effect of diabetes on ABR recordings. These changes included an elevation in the hearing threshold and increased wave latency. Also, a decrease in the amplitude with changes in waveforms was observed.27,28

Our study showed that eight weeks’ treatment of diabetic rats with folic acid at different doses (40 and 80 mg/kg) did not significantly improve the hearing threshold and waves II and V latency compared to control diabetic rats. There are some differences in rat ABR compared to humans that we have considered in this study. It seems wave II generated in the cochlear nuclear complex is the most prominent in rat ABR recordings.29

Morphological changes in DAN mainly include atrophy of stria vascularis, thickening of the basilar membrane in the stria vascularis, and decrease in the number of spiral ganglion neurons.7,30

Similar to other studies, the histological changes shown in our study following the induction of diabetes in rats included a decrease in the volume of the stria vascularis, spiral ganglion, spiral ligament and a reduction of spiral ganglion neurons. The stereological method we used in this study allowed us to examine structural and cellular changes in DAN more quantitatively and accurately. The structural deficits in DAN are consistent with the functional findings of ABR. In this study, the preventive effect of folic acid on structural changes at different doses (40 and 80 mg/kg) was not shown.

Our study revealed a significant decrease in HCY levels following folic acid administration in diabetic rats. This reduction is more significant at higher doses of folic acid (80 mg/kg). Its role and underlying mechanism in developing vascular complications in diabetes and reducing its incidence rates after treatment are not precisely defined.22,31

The increased oxidative degradation of nitric oxide (a regulator of endothelial homeostasis) during oxidative stress is considered the presumptive mechanism of endothelial vascular injury in elevated HCY levels.32,33 Through its bioactive form, 5-methyltetrahydrofolate, folic acid donates a methyl-group for HCY remethylation to methionine.17,34

Our study showed that plasma MDA levels were significantly increased following folic acid treatment in diabetic rats. This finding is consistent with other findings in experimental studies on the folic acid treatment of neuropathy.10 Another important finding in this study was the lack of SOD increase following folic acid treatment. These antioxidant enzymes control and eliminate ROS during oxidative stress in cells.35 Based on the findings of this study, folic acid lowered plasma HCY and MDA levels significantly, while it had no significant effect on SOD. It may imply that oxidative stress is reduced via a mechanism other than an increase in the amount of action of SOD.

Another perception drawn from these findings is that, due to the lack of improvement in structural and functional disorders in DAN and the lack of significant change in SOD levels, SOD may be a better measure of oxidative stress than HCY and MDA.

It can be deduced that the selected doses of folic acids were not high enough to produce meaningful action on diabetic neuropathy. However, based on our scientific bibliography, the selected doses were higher than those applied in similar studies. Also, one limitation of this study was the short duration of treatment in the DAN rat model. It is suggested to evaluate the therapeutic effects of folic acid for a longer time in the animal model.

Materials and Methods

Materials

Folic acid and Streptozotocin (STZ) were purchased from Sigma-Aldrich Chemical Company (Steinheim, Germany).

Animals Preparation

Thirty-two male Sprague–Dawley rats of similar age in the weight range of 250–300 g were obtained from the Center of Comparative and Experimental Medicine (Shiraz, Iran). The rats were kept under standard lighting (12-h-light/dark cycles), humidity (25–35%), temperature (22–26 °C) per day and had free access to food and water. The rats were randomly allocated to four groups (eight animals in each group). They included non-diabetic rats with distilled water (normal group), type 1 diabetic rats with distilled water (DM group), and type 1 diabetic rats that received folic acid 40 mg/kg/day (DM + FA40) and 80 mg/kg/day (DM + FA80).

The experimental procedures were approved by the Ethics Committee of Shiraz University of Medical Sciences (Ethics code: IR.SUMS.REC.1397.459).

Induction of Type 1 Diabetes Mellitus

A single intraperitoneal administration of STZ (60 mg/kg) was dissolved in cold citrate buffer and injected into animals to induce type 1 DM. Three days after induction, fasting blood sugar (FBS) levels were checked using a Glucometer (Accu-check, Germany), and rats with FBS levels greater than 300 mg/dL were considered type 1 diabetic animals.36 The confirmation day was considered 3 days after STZ injection.

Treatment

After the confirmation of diabetes, two groups received an oral solution of folic acid 40 mg/kg/day (DM + FA40) and 80 mg/kg/day (DM + FA80) by gavage once a day for eight weeks. Normal and diabetic groups received an equal amount of distilled water.

After eight weeks of treatment, rats were prepared for auditory assessment by ABR and histological studies.

ABR Assessment

At the end of treatments, xylazine (10 mg/kg) and ketamine (75–100 mg/kg) were administered to the rats via intraperitoneal injection.9 A thermal blanket and heat lamp were used to avoid hypothermia during anesthesia. In the ABR recording, the ground electrode (ECG Ambu Blue Sensor, Penang, Malaysia) was fixed on the dorsal neck of the rat. Also, the active electrode (+) and the reference electrode (−) were attached to the forehead and postauricular area of the right and left ears, respectively. Evoked potentials were achieved by intra-acoustic EP25 (Copenhagen, Denmark) system with OtoAccess software (Middelfart, Denmark) after releasing clicks to the right ear via embedded earphones. Minimum 700 stimuli at a rate of 11.1 Hz were distributed to the right ear, and electrode impedance was below 3 KΩ. ABR evaluations were accomplished using a 100–3000 Hz band pass filter and a time window of 10 ms.37 Evoked potentials were estimated according to the following parameters: absolute latencies and wave morphology of waves II and V and hearing threshold. The hearing threshold was detected in 5 dB steps declining from the maximum stimulus of 70 dB until wave patterns morphology disappeared.

Tissue Preparation and Stereological Analysis

Preparation of Inner Ear in Rat

At the end of the study, all animals were sacrificed after induction of deep anesthesia by intraperitoneal injection of 100 mg/kg ketamine and 10 mg/kg xylazine.9 The temporal bone was carefully dissected and separated from skull bones, and decalcification of the temporal bone was performed using 8% HCL and 8% formic acid for 3 days.38 All temporal bones underwent tissue processing and were embedded in paraffin wax. The paraffin blocks were cut into a 25 μm thickness serial section and the external acoustic meatus axis by a microtome (Microme, Germany). All of the serial sections of the cochlea were collected and separated from apical to basal cochlear.

Thirty-two to thirty-six sections of cochlear tissue per rat were obtained, and all slides were stained with hematoxylin and eosin. Afterward, 8–12 sections of the 32–36 sections of the cochlea in a systematic uniform random sampling were selected. The first section was randomly selected, and the following sections were chosen at equal intervals.

Stereological techniques were applied to estimate the volumes of the spiral ganglion, spiral ligament, and stria vascularis and the number of neurons in the spiral ganglion. The cochlear section evaluation was done by an examiner blind to the animal groups.

Estimation of the Spiral Ganglion, Spiral Ligament, and Stria Vascularis Volumes

The live figure of each cochlea section was evaluated by a video microscopy system and stereology software (StereoLite, SUMS, Shiraz, Iran).

Regions of the selected structure (spiral ganglion, spiral ligament, and stria vascularis) were recognized in each cochlea section (Figure 4a). The volume of the selected structure was analyzed by the point-counting methods based on “Cavalieri’s principle” at the final magnification of 20 on 8–12 sections per animal.39−41

Figure 4.

Figure 4

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Stereological methods estimate the stria vascularis, spiral ligament, and spiral ganglion volumes. The stria vascularis, spiral ligament, and spiral ganglion of the cochlea are displayed in the histological section (a). The volume of the stria vascularis, spiral ligament, and spiral ganglion was evaluated by Cavalieri’s principle and point-counting technique (b).

The probe of stereology (a grid of points) was overlaid on the cochlear images (Figure 4b), and the stereology software calculated the cross-sectional area of the selected structure “∑A.” Then, the cross-sectional area of the selected structure “∑A” was multiplied by the distance between the sections (T). The following formula estimated the volume of the chosen structure:

graphic file with name pt2c00140_m001.jpg

where V is the volume of the selected structure, T is the distance between two selected sections, a/p is area per point, and ∑P is the total points hitting the chosen structure. The area per point (a/p) was 722,500 (850 × 850) μm2 and an average of 121 points were counted per animal.

Estimation of the Neuron Numbers in the Spiral Ganglion

The spiral ganglion is a collection of bipolar neuron cell bodies in the modiolus of the inner ear, the lemon-like shaped central axis of the cochlea whose fibers innervate the Corti organ.39 The optical dissector is one of the stereological techniques for counting spiral ganglion neurons in a histological section thickness.41−43

The site of the microscopic field was chosen by moving the stage in directions (x and y) at the same distances according to systematic uniform random sampling order. An objective lens (40×, NA 1.30) was employed by oil immersion.

The optical disector is made of several parts. An Eclipse microscope (E200, Nikon, Tokyo, Japan) with a large numerical aperture (NA = 1.30) × 40 oil-immersion objective was linked to a video camera, which conveyed the live image of the microscope to a computer monitor. An electronic microcator with a digital readout (MT12, Heidenhain, Traunreut, Germany) for estimating the movements in the z-axis with a precision of 0.5 μm.

One of the stereological probes is an unbiased counting frame employed to count the number of select cells by a stereology software system (Stereolite, SUMS, Shiraz, Iran). The unbiased counting frame, composed of two exclusion lines (the lower and left borders and their extensions) and two inclusion lines (the upper and right borders), was overlaid on the live image at a final magnification of 1500× (Figure 5a).

Figure 5.

Figure 5

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Stereological technique estimation of the numbers of spiral ganglion cells. Estimated number of ganglion cells assessed using the optical dissector method. The cell nuclei that appeared in the high focus during scanning of the height of the disector were counted (the arrow) (a and b).

The guard areas are at the superior and inferior parts of the histological sections. These regions were applied to avoid cutting tissue artifacts that occur throughout tissue processing in these areas of the sections. Any counting incident in focus within the up (the first 3.5 μm) or down guard zones was deleted. Each nucleolus cell that came into focus within the guard areas was not counted. The “height of disector” was the distance between the guard areas, 18 μm here. Every nucleolus cell appearing in focus inside the later focal sampling plane was selected if it was located completely or partially inside the counting frame (Figure 5b) and did not contact the forbidden lines.41−43

The numerical density (NV) of the spiral ganglion neurons was evaluated using the following formula:

graphic file with name pt2c00140_m002.jpg

where “∑Q̅” is the number of the spiral ganglion neurons coming into high-quality focus in disector height, “ΣP” is the total of the unbiased counting frames in all fields of microscopic, and “h” is the height disector; “a(f)” is the area of counting frame, “t” is the true section thickness calculated with the microcator, and “BA” is the microtome block advance set. The thickness of the internal ear section was calculated in the whole microscopic fields of view with uniform random sampling order from each section.

On average, 100–200 neurons were counted in 26 disectors in 8–12 sections per animal. The area of the counting frame (a(f)) was 1082.41 (32.9 × 32.9) μm2. The actual average thickness of the sections was 22 μm (t), and “BA” was 25 μm. The number of neurons was estimated by multiplying the NV by V (submucosa or muscularis layers).

Biochemical Assays

At the end of treatments, SOD activity, MDA serum levels, and serum activity of HCY were measured in all groups. We used the SOD ELISA kit (Cayman, Ann Arbor, MI, USA), Rat MDA ELISA kit (CusabioBiotech, China), and HCY ELISA kit (Axis Homocysteine Enzyme Immunoassay, IBL, Germany), respectively.

Statistical Analysis

Data were presented as mean for stereological results and mean ± SEM for other results. Statistical analysis was determined using one-way analysis of variance (ANOVA) for parametric data such as latency of wave II, hearing threshold, the volume of spiral ganglion, spiral ligament, and stria vascularis, number of spiral ganglion neurons, SOD, MDA, and HCY. Also, the Kruskal–Wallis test was applied for nonparametric data, such as different forms of wave V. GraphPad Prism software (Version 6) was used to compare data. If a significant difference was obtained, Tukey’s or Dunn’s post hoc test located the source of the difference. P < 0.05 was considered statistically significant.

Conclusions

DAN is one of the severe complications of diabetes that seriously impacts the quality of life. Our study showed that folic acid is ineffective in relieving structural and functional disorders in DAN, despite its effectiveness in reducing HCY and MDA. Due to this issue, more studies are needed to find potent drugs for preventing and treating this complication in diabetic patients.

Acknowledgments

This study was financially supported by Shiraz University of Medical Sciences (Grant no. 16690). The authors would like to thank Shiraz University of Medical Sciences, Shiraz, Iran, and Ms. Sheryl Thomas-Nikpoor, English language editor, for her valuable comments in editing this manuscript.

Author Contributions

A.D., H.M., and K.I. conceived and designed the research. A.D., K.I., S.K.-D., A.D., E.N., and F.P. conducted experiments. A.D., S.K.-D., A.D., E.N., and F.P. contributed new reagents or analytical tools. A.D., H.M., E.N., and F.P. analyzed the data. A.D. and K.I. wrote the manuscript. All authors read and approved the manuscript.

The authors declare no competing financial interest.

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