2026 Volume 51 Issue 5 Pages 295-302
Humans rely heavily on visual function to gather information, and loss of vision has a significant impact on quality of life. However, it is difficult to quantitatively evaluate the effects of chemical compounds on visual function in toxicity studies using animals (such as OECD guideline studies). Consequently, evaluation including ophthalmological and histopathological examinations has played a major role to date. Visually evoked potential (VEP) is a type of brain wave that reflects the activity of the entire visual pathway, including the retina, optic nerve, and visual cortex. We investigated whether VEP could detect the effects of acrylamide, a toxicant known to affect peripheral nerves, on visual function. Acrylamide was administered to rats via drinking water at concentrations of 0 (control) and 200 ppm for 4 weeks, and electroretinograms (ERGs) and VEPs were recorded at weeks 0, 2, and 4. After the 4-week treatment period, the eyes and optic nerves were examined by light microscope. Acrylamide exposure significantly delayed VEP latency, while no effects were observed on the retina and optic nerve by ERG or histopathology. A significant decrease in grip strength in the hindlimbs and degeneration of sciatic nerve fibers were observed in the acrylamide-treated group, indicating that acrylamide damaged peripheral nerves. In conclusion, our study demonstrated that VEP can detect the effects of acrylamide on visual function earlier than histopathological examination, suggesting that VEP could be useful for detecting early-phase effects of chemical compounds on visual function and for evaluating whether morphological changes observed in toxicity studies are toxicologically significant.
Loss of vision has a significant impact on the quality of life in humans (Onodera et al., 2015). The ocular toxicity of chemical compounds, including pharmaceutical drugs, pesticides, and industrial chemicals, is carefully assessed using experimental animals before these compounds are released to the market. It is, however, difficult to objectively evaluate the effects of chemical compounds on visual function in toxicity studies using animals. Therefore, morphological assessments such as ophthalmological examinations (using slit-lamp microscopy and fundoscopy) and histopathological examinations have played a major role in evaluating the effects on visual function to date (ICH, 1998; OECD, 2018). A key issue is that morphological findings and visual functional changes might not necessarily correlate with each other. There are cases where morphological changes are not accompanied with alterations in visual function, and vice versa. For example, an early phase of diabetic retinopathy in humans can be detected by fundoscopy but remains asymptomatic (Wong et al., 2016). On the other hand, levofloxacin has been shown to cause retinal dysfunction in rats without any detectable morphological changes in the retina (Nomura et al., 1992). Therefore, it is important to evaluate not only morphological changes but also visual function.
The electroretinogram (ERG) is an electrical potential generated by the retina in response to light and detected at the surface of the cornea (Brandli and Stone, 2015; Robson et al., 2022). It has been widely used to objectively evaluate retinal function not only in humans but also in experimental and companion animals. Although commonly used and procedurally standardized (Robson et al., 2022), the ERG serves solely as an indicator of retinal function. Therefore, the ERG cannot detect abnormalities in the optic nerves, optic chiasm, or visual cortex.
Visually evoked potential (VEP) is a kind of electroencephalogram that reflects activity throughout the entire visual pathway, including the retina, optic nerve, optic chiasm, and visual cortex (Ridder and Nusinowitz, 2006; Onodera et al., 2015). VEP is considered a valid method for objectively evaluating visual function and has been clinically used in humans (Zheng et al., 2020). VEP’s amplitude (the magnitude of cortical response) and latency (the period of time from light stimulation to cortical response) are usually evaluated, but it is reported that the VEP latency is strongly associated with better visual acuity and that it is the most reliable parameter for the evaluation of the integrity of the visual pathway in humans (Lenassi et al., 2008). It has also been recorded in animals such as mice, rats, dogs, and monkeys (Weinstein, 1977; Strain et al., 1990; Sasaki et al., 2003; Ridder and Nusinowitz, 2006). However, reports evaluating the effects of chemical compounds on VEP in rodents are limited, and, to our knowledge, no report has compared the sensitivity of VEP with that of conventional methods (i.e., histopathology) in detecting toxic effects on the eyes.
Acrylamide is a chemical compound known to affect peripheral nerves as well as optic nerves (Vidyasagar, 1981). It has been reported that acrylamide treatment (30 mg/kg, 5 days per week for 4 weeks) in rats causes impairment of axonal transport in the optic nerves, which is considered an important factor in the pathogenesis of axonal degeneration in acrylamide neuropathy (Sabri and Spencer, 1990). This finding suggests that histopathology may be the most sensitive morphological analysis for detecting acrylamide-induced ocular toxicity. In addition, acrylamide (10 mg/kg, 5 days per week for 6-10 weeks) has also been shown to affect VEP in macaque monkeys (Merigan et al., 1982). However, acrylamide does not affect the retina (Fox and Boyes, 2019). Therefore, we selected acrylamide as an inducer of morphological and functional changes in the optic nerves without affecting other parts of the eye, such as the retina.
The aim of this study was firstly to demonstrate that VEP can detect impairment of visual function in rats caused by acrylamide, and secondly to examine whether, in rats treated with acrylamide, VEP can detect alterations in the optic nerves earlier than histopathology by light microscope, which is usually used in guideline toxicity studies.
Acrylamide (for electrophoresis, purity: 100.0%) was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).
Animals and husbandryAll the animal experiments were performed in accordance with The Guide for Animal Care and Use of Sumitomo Chemical Company, Ltd., and were reviewed and approved by the Institutional Animal Care and Use Committee. Seven-week-old female RccHan:WIST rats were obtained from Japan SLC, Inc. (Shizuoka, Japan). Following a 7-day quarantine period, animals in a healthy condition received a surgically placed electrode implant as described in subsection F). After the surgery, the animals which showed normal ERG and VEP responses were randomly assigned to the control or acrylamide groups. The animals were housed in a clean animal room. During the study, the environmental conditions in the animal room were set to maintain a targeted temperature range of 22-26°C and a relative humidity range of 40–70%, with frequent ventilation (more than 10 times per hour) and a 12-hr light (8:00–20:00)/ 12-hr dark (20:00–8:00) illumination cycle (except for the dark adaptation period before ERG and VEP recordings). A commercially available pelleted diet sterilized by 60Co (30 kGy) irradiation (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and deionized water (with or without acrylamide) were provided ad libitum throughout the study.
Dosing proceduresAt age 11 weeks old, rats were administered acrylamide dissolved in deionized water at 200 ppm via drinking water (the acrylamide group; n=8) for 4 weeks or offered deionized water in the same way (the control group; n=8). The dosing formulation was prepared once every two or three days and used for dosing immediately after preparation. Water bottles were exchanged for new ones at every preparation. Acrylamide was considered stable in water at room temperature based on a previous report (Maronpot et al., 2015).
Observation itemsClinical signs (daily), body weight (BW, once a week), water consumption (twice a week), grip strength (Week 4), ERG (Week -1, 2 and 4), VEP (Week -1, 2 and 4), and histopathology (Week 4, on eye, retina, optic nerve, sciatic nerve) were recorded. Daily water consumption was calculated on each animal by weighing water bottle before and after each measurement period. Acrylamide intake at each measurement point was calculated based on daily water consumption and most recent body weight, and the overall acrylamide intake (mg/kg/day) was derived from the simple average of acrylamide intake at each measurement point.
Grip strengthForelimb and hindlimb grip strength were measured using a small animal dynamometer (GPM-100, Melquest, Toyama, Japan). Briefly, an animal was allowed to grip a T-shaped grip bar with its fore paws or hind paws, and was pulled back gently until it releases the bar. The average of two valid grip strength measurements (in kilograms) was used for evaluation.
Electrode implant surgeryThe method used for electrode implantation in rodents in this study was previously published (Sasaki et al., 2003; Sasaki et al., 2004; Tomita et al., 2009; You et al., 2015). Briefly, the animals underwent surgery when they were 8 weeks old. During the surgery, rats were anesthetized with isoflurane and their body temperature was maintained at 37°C, and ophthalmic ointment (Ecolicin ophthalmic ointment, Santen Pharmaceutical Co., Ltd., Japan) was applied to both eyes to prevent corneal dryness. The skin of the surgical area was shaved and disinfected with iodine, and a longitudinal skin incision on the midline of the head skin was made. Two small holes were drilled into the skull using a hand drill, 7 mm behind the bregma and 3 mm lateral to the midline (on both sides). A screw electrode was implanted through each hole into the visual cortex to a depth of approximately 0.5 mm (for the positive electrodes). A third hole was drilled on the midline 3 mm rostral to the bregma, and a screw electrode (for the negative electrode) was implanted through this hole. Dental cement was applied to secure the screws. The incised skin was sutured, and the rats were given meloxicam (Metacam, Boehringer Ingelheim Animal Health Japan Co., Ltd., Japan) for at least three days. VEP recording was initiated at least 10 days after the surgery, and clinical signs were carefully observed every day and BW was frequently measured during this period.
ERG and VEP recordingERG and VEP recordings were performed on the same day using a Ganzfeld dome and Neuropack S1 (MEB-9400, NIHON KOHDEN, Tokyo, Japan). Before recording the ERG and VEP, the rats were placed in a dark room overnight (dark adaptation), and a weak red light was used while recording. During the recordings, rats were anesthetized with isoflurane, body temperature was maintained at 37°C, and drops of a mydriatic agent (Mydrin-P, Santen Pharmaceutical Co., Ltd., Japan) and local anesthetic (Benoxil, Santen Pharmaceutical Co., Ltd., Japan) were administered to both eyes.
For the ERG recording, the positive electrodes (contact lenses) were placed on both eyes, and the reference electrode was positioned on the tongue. The ERG was recorded using white flashes at intensities of 0.01 candelas (cds)/m2 (0.5 Hz, 32 responses averaged) and 1 cds/m2 (0.5 Hz, 16 responses averaged).
Immediately following the completion of the ERG recording, the VEP consisting of three waves (P1, N1, and P2) was recorded using white flashes at intensities of 0.1, 1, or 10 cds/m2 (1 Hz, 200 responses averaged). The N1 amplitude and latency were evaluated as previously described (Sasaki et al., 2003; Sasaki et al., 2004; Tomita et al., 2009; You et al., 2015). Ambient white noise was present during the VEP recording in order to prevent any interference by auditory evoked potentials.
HistopathologyAt the end of the dosing period, six out of eight animals in each group were euthanized under isoflurane (ISOFLURANE Inhalation solution, Mylan Inc., Tokyo, Japan) anesthesia by perfusing a mixed fixative solution of 2.5% glutaraldehyde and 2% paraformaldehyde into the circulatory system via the heart. Eyes (including retina), optic nerves, and sciatic nerves were removed and immersed in 10% neutral buffered formalin. Paraffin-embedded sections were prepared from the eyes (bilateral), an optic nerve (unilateral), and a sciatic nerve (unilateral), and then stained with hematoxylin and eosin (HE) for histopathological examination. In addition, for three of these six perfusion-fixed animals, the optic nerves and sciatic nerves on the contralateral side were embedded in epoxy resin and stained with toluidine blue. For the remaining two animals in each group, blood was collected from the abdominal aorta under isoflurane anesthesia prior to euthanasia, and the above-mentioned organs were removed. The optic nerves and sciatic nerves were immersed in 10% neutral buffered formalin, and the eyes were fixed in Davidson’s solution, subsequently preserved in 10% neutral-buffered formalin, and finally stained with HE.
Statistical analysisFor comparison between the control and acrylamide groups, the F-test was applied. If the variance was homogeneous, the Student t-test was used. If the variance was heterogeneous, the Aspin-Welch-test was used. When comparing VEP amplitude and latency at Weeks 2 and 4 with those at Week -1, a paired-t-test was used. Each analysis was conducted by 2-tailed tests at the 0.05 and 0.01 significance levels.
Abnormal gait (impairment of hindlimb use) was observed in one animal in the acrylamide group at Weeks 2 and 3, and in all the animals in the acrylamide group at Week 4. No other abnormal findings were observed in the acrylamide group.
BW and water consumptionBW and water consumption were significantly decreased in the acrylamide group. The averaged daily acrylamide intake was 23.1 mg/kg/day.
Grip strengthIn the acrylamide group, the grip strength was significantly decreased in the hindlimbs but not affected in the forelimbs (Fig. 1).
Grip strength. Grip strength of rats treated with acrylamide (200 ppm in drinking water) for 4 weeks. Data are expressed as mean ± S.D. (n=8/group). ** indicates statistical significance from the control (p<0.01).
Both HE and toluidine blue staining revealed degenerated nerve fibers in the sciatic nerves in rats treated with acrylamide for 4 weeks but no abnormal findings in the optic nerves (Fig. 2). The eyes, including the retina, showed no abnormal findings related to acrylamide treatment (data not shown). Since the rats were treated with acrylamide continuously for 4 weeks, no histopathological findings at 4 weeks suggested that there would be no abnormal findings earlier than 4 weeks. Dysplasia in retina and mineralization in cornea were observed in some rats, but they were considered not affecting the overall outcome of this study because they were known as spontaneous findings in rats and observed in both the control and acrylamide groups at similar incidences in this study.
Histopathology of sciatic nerves and optic nerves. Sciatic nerves (top) and optic nerves (bottom) were stained with hematoxylin-eosin (HE) and toluidine blue and examined by light microscope. All images were obtained from perfusion-fixed samples. Degenerated nerve fibers (arrowhead) were observed in sciatic nerves of acrylamide-treated rats.
There were no abnormalities in the ERG at 0.01 or 1 cds/m2. Typical ERG recordings from an animal in the acrylamide group before treatment and at Week 4 are shown in Fig. 3.
Typical examples of electroretinograms (ERGs). Typical examples of ERGs from a rat in the acrylamide (200 ppm in drinking water) group before (Week -1) and after (Week 4) treatment. The arrows indicate flash stimulation (1 cds/m2).
At Week -1 (before the initiation of dosing), we confirmed that the electroencephalogram signals recorded from the implanted electrodes corresponded to visual stimuli (Fig. 4). The amplitude and latency of the recorded potentials were increased and shortened, respectively, as the intensity of the stimulus light was strengthened. In addition, when one eye was completely covered, the amplitudes and latencies of the potentials recorded from the corresponding hemisphere were markedly decreased and delayed.
Visually evoked potential (VEP) recorded from non-treated rats. (A) Typical examples of VEP from a non-treated rat when both eyes or a right eye were stimulated. The arrows indicate flash stimulation (10 cds/m2). (B) The mean of N1 amplitude and latency with or without flash stimulation on each eye, or when the intensity of flash stimulation was changed. Data are expressed as mean ± S.D. (n=8/group). *,**: statistically significant at p<0.05 and 0.01 (paired t-test).
Typical examples of VEPs before and after treatment are shown in Fig. 5, and the amplitude and latency of VEPs at Weeks -1, 2, and 4 are summarized in Fig. 6. The latency was statistically significantly delayed at Week 2 (left hemisphere) and Week 4 (left and right hemispheres) compared to values in the concurrent control group and initial values in the acrylamide group (before treatment). The latency of the VEP of the right hemisphere at Week 2 did not reach statistical significance, but the value was higher than the value in the concurrent control and the initial value in the acrylamide group, and therefore, it was considered to be increased by acrylamide.
Typical examples of visually evoked potential (VEP) of an acrylamide-treated rat. Typical examples of VEP from an acrylamide (200 ppm in drinking water) -treated rat before (Week -1) and after (Week 4) treatment. The arrows indicate flash stimulation (10 cds/m2).
Effects of acrylamide on amplitude and latency of visually evoked potential (VEP). The mean of N1 latency and amplitude from the control and acrylamide (200 ppm in drinking water) groups at Week (W) -1, 2, and 4. Data are expressed as mean ± S.D. (n=8/group). *,**: statistically significant at p<0.05 and 0.01 compared to the control group (Student t test). $, $$: statistically significant at p<0.05 and 0.01 compared to each value at Week -1 (paired t-test). VEP was recorded with flashes at 10 cds/m2.
The amplitude was not affected at either Week 2 or 4 by acrylamide under the conditions of this study.
Acrylamide is a well-known toxicant affecting peripheral nerves (Vidyasagar, 1981; Fox and Boyes, 2019), and it has been reported that acrylamide-treatment at 30 mg/kg/day for 4 weeks in rats with impaired optic nerve function (Sabri and Spencer, 1990). In this study, acrylamide was dosed to rats via drinking water for 4 weeks, with an average intake of 23.1 mg/kg/day, which was comparable to the previously reported dosing levels. In fact, rats showed impairment of hindlimb use and decreased grip strength at Week 4. Histopathology showed degenerated nerve fibers in the sciatic nerves. These findings indicate that the dose level used in this study was sufficient to affect peripheral nerves in the rats.
The amplitude and latency of the potentials increased and shortened, respectively, as the intensity of stimulus light was strengthened. This clearly showed that the potentials recorded from the brain cortex were related to light stimulation. In addition, when one eye was completely covered, the amplitudes and latencies of the potentials recorded from the corresponding hemisphere were markedly decreased and delayed. Based on these results, we concluded that the potentials recorded from the brain cortex were induced by light stimulation and thus represented VEP. Since the VEP changed depending on the condition of visual stimulation, VEP is considered useful for evaluating the effects of chemical compounds on visual function in rats.
There were no abnormal histopathology findings in the retina (data not shown), and there was no effect on ERG by acrylamide treatment. Therefore, it was considered that the retina was neither morphologically nor functionally affected by acrylamide under the conditions of this study, which is consistent with the previous findings (Fox and Boyes, 2019).
Histopathology revealed no abnormalities in the optic nerves after the 4-week treatment with acrylamide, but VEP was affected. The N1 latency of VEP was statistically significantly delayed at Week 4. Although the N1 latency of VEP recorded from the right hemisphere did not reach statistical significance at Week 2, it was considered that the latency of VEP was affected by acrylamide, because the mean value was greater than both the pre-treatment and concurrent control values, and the latency of VEP from the left hemisphere reached statistical significance. VEP earlier than Week 2 was not evaluated in this study, but it was considered that it would be difficult to detect acrylamide’s effects on VEP earlier than Week 2, since VEP from only one side reached statistical significance even at Week 2. The N1 amplitude was not changed in this study, and the reason for the delayed N1 latency without a change in amplitude remains unclear. Since N1 amplitude depended on the intensity of light stimulation (Fig. 4B), it could be affected by chemical compound exposure if dosing is continued for a longer period and pathological changes in the optic nerves progress. However, under the conditions of this study where there was no histopathological finding in the optic nerves, N1 latency was more sensitive than N1 amplitude. This is consistent with the report in humans that the VEP latency is strongly associated with better visual acuity and that it is the most reliable parameter for the evaluation of the integrity of the visual pathway in humans (Lenassi et al., 2008).
Since VEP but not histopathology detected the effects of acrylamide on visual function in rats, it is possible that VEP is more sensitive for detecting the effects of chemical compounds on the eyes at an early phase of treatment. Further investigation is needed to determine if this is true for other chemical compounds. In addition, it wasn’t clarified in this study how the alterations in VEP by acrylamide affected vision in the rats (i.e. a significant impact of vision, or asymptomatic), and further research is necessary to clear it, too.
Overall, VEP was able to detect the effects of acrylamide on visual function in rats at an early stage of treatment, before morphological changes in the optic nerves occurred. VEP can be useful for evaluating the toxicological impact of morphological changes on visual function, as well as short-term screening studies.
We thank contributors to this research project from Sumitomo Chemical Company, Ltd. We also would like to thank ASCA Corporation who provided proofreading services funded by Sumitomo Chemical Company, Ltd. We acknowledge the support of ASCA Corporation in the editing of a draft of this manuscript.
FundingNo funding was provided for the work.
Conflict of interestThe authors are employed by Sumitomo Chemical Company, Ltd.
Data availabilityThe data that support the findings of this study are confidential and not openly available. Data are located in controlled access data storage at Sumitomo Chemical Company, Ltd.
Author contributionsConceptualization: KK, HA
Funding acquisition: SF, HA
Data acquisition, analysis and interpretation: KK, YS, KO, SF
Writing – original draft: KK, YS
Writing – review & editing: SF, HA
Ethical approval and consent to participateNot applicable.
Patient consent for publicationNot applicable.
