Anxiety-like behaviors increased in juvenile rats in the streptozotocin-induced diabetes model during the early stage

Abstract

BACKGROUND

Diabetes is an endocrinopathy characterized by the inability to produce insulin or insufficient insulin production because of the destruction of beta cells in the pancreas caused by autoimmune or other epidemiological factors. Diabetes can lead to physical and psychological complications.

AIM

To investigate early behavioral changes in an experimental diabetes model in juvenile rats.

METHODS

In this study, a total of 14 juvenile male Wistar rats (7 rats per group) were used. To induce the diabetes model, 120 mg/kg streptozotocin (STZ) was administered, while the control group received 1 mL/kg physiological saline. The open field test and the hole-board test were performed for five minutes each. Unpaired comparisons were evaluated using Student’s t test or the Mann-Whitney U test. Correlations were analyzed using the Pearson test.

RESULTS

Severe hyperglycemia was observed in rats in the diabetes mellitus model induced by STZ, and all diabetic rats displayed glucose levels above 400 mg/dL. In the open-field test, the time spent in the central area, the number of rearings, and the total distance traveled were significantly lower in diabetic rats than in naive rats (P < 0.05). In the hole-board test, the duration and frequency of total head dipping were considerably lower in diabetic rats than in healthy rats (P < 0.01). The grooming time (P < 0.01) and freezing time (P < 0.0001) increased dramatically in the diabetes mellitus group. A remarkable correlation was found between behavioral changes and hyperglycemia.

CONCLUSION

Our data indicate that early-stage diabetes induced by STZ increases anxiety-like behaviors and impairs exploratory behaviors. These behavioral changes may also occur in children with undiagnosed or untreated hyperglycemia and can provide important clues in clinical practice.

Key Words: Anxiety-like behaviors; Diabetes mellitus; Early-stage changes; Hole-board test; Juvenile rats; Open field test; Streptozotocin

Core Tip: In this study, the relationship between hyperglycemia and anxiety-like behaviors in a streptozotocin-induced diabetes model was investigated in juvenile rats. An increase in anxiety-like behaviors and a decrease in exploratory behaviors were observed in juvenile rats during the early stages of diabetes. Both behavioral changes were correlated with hyperglycemia.

INTRODUCTION

Diabetes mellitus (DM) is a group of metabolic disorders characterized by hyperglycemia emerging from defects in insulin secretion, insulin action, or both[1]. DM leads to hyperglycemia because of insufficient glucose utilization as an energy source and excessive glucose production caused by inappropriate gluconeogenesis and glycogenolysis[2,3]. The increase in the prevalence of DM, one of the most common metabolic disorders, is a cause for concern, particularly on a global scale[4]. The global prevalence of diabetes is estimated to be 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045[5].

DM can deteriorate different organ systems[6]. DM patients have a considerably increased risk of cardiovascular disease, heart failure, chronic kidney disease, fatty liver disease, and eye and foot diseases[7]. Furthermore, DM is significantly correlated with and associated with several neurological and psychiatric disorders[8].

In their meta-analysis, Smith et al[9] reported that diabetes is associated with anxiety disorders and high anxiety symptoms. Furthermore, anxiety-like behaviors have been reported to increase in streptozotocin (STZ)-induced diabetes models[10]. Preclinical studies are based on adult rodents and the late-stage effects of DM[10-13]. According to these studies, adult rats exhibit anxiety-like behaviors, particularly in the elevated plus maze test[10]. Caliskan et al[10] reported that freezing and postural changes associated with anxiety increased specifically in diabetic rats in the elevated plus maze. Furthermore, late-stage effects observed in rats in a DM model include increased depressive behavior, cognitive impairment, and neurological damage, such as neuropathic pain[14-16].

Studies presented in the literature have been conducted in late-stage and adult rats. Early diagnosis of diabetes is very important, as with many other diseases. Behavioral and molecular changes that occur in the body during the early stages can inform diagnosis and treatment. Therefore, there is a need for diabetes studies in young groups and for examining the effects of early-stage diabetes. In this regard, our aim is to examine the effects of hyperglycemia during the early stages of STZ-induced diabetes in juvenile rats on exploratory behavior, anxiety, and locomotor activity.

MATERIALS AND METHODS

Animals

This study involved 14 juvenile Wistar albino male rats over a period of 21 days. The rats were purchased from the Laboratory Animal Production Laboratory at Balıkesir University. This study was conducted with the approval of the Institutional Animal Care and Use Committee of Balıkesir University, approval No. 2025-5/27. They underwent a one-week adaptation period. The animals were kept in a house on a 12-hour light/dark cycle at a constant temperature of 22 ± 2 °C and a humidity of 50% ± 5%. The feeding regimen was ad libitum, and the rats had access to food and water throughout the experiment. For induction of the diabetes model, the rats were fasted for eight hours before STZ injection. The Guide for the Care and Use of Laboratory Animals, published by the United States National Institutes of Health, was used as a reference for the handling of the rats[17]. This study was also conducted in accordance with the ARRIVE guidelines 2.0. (Animal Research: Reporting of In Vivo Experiments)[18].

STZ treatment

STZ is frequently used to induce diabetes in experimental models[19]. STZ was administered at 120 mg/kg intraperitoneally (i.p.)[20-24]. The control group received 1 mL/kg saline. STZ was dissolved in citrate buffer at pH 4.5. The rats were fasted for eight hours the night before STZ injection. Twenty-four hours after STZ administration, one drop of blood was collected from the tail vein of each rat to confirm the presence of DM, and body weight was measured. Similar weighing and blood glucose measurements were performed on the control group rats. A blood glucose level ≥ 300 mg/dL (16.6 mmol/L) was considered to indicate diabetes. Blood glucose and weight measurements were performed on the rats’ 30^th birthday. A glucometer (Lifechek TD-4287) was used to measure glucose levels. A disposable lancet strip was used to draw a drop of blood from the rats' tails. In addition, to prevent the rats from being adversely affected by this procedure, an ointment containing 5% lidocaine (50 mg of lidocaine per gram) was applied to the tail site from which blood was drawn, after which one drop of blood was collected. After the blood was measured, the tail was wiped with alcohol-soaked cotton. Blood glucose levels were measured on days 30, 31, and 32 for all the rats (Figure 1). The rats were euthanized using ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg i.p.).

Figure 1 Experimental timeline. STZ: Streptozotocin.

Open field test

The open field test (OFT) is used to assess general locomotor activity and anxiety-like behaviors[25]. The open field and new environment induce anxiety-like behaviors. Increased anxiety causes rats to spend time near the walls of the open field. As anxiety decreases, rats spend more time in the center and exhibit rearing behavior. A square open field apparatus measuring 60 cm × 60 cm and 50 cm high was used. The total distance traveled and the time spent in the center, rearing, freezing and grooming were recorded for five minutes. The experimental room was kept quiet under constant lighting (110 Lux, warm light). A camera recorded the test, and the tapes were analyzed blindly. The researchers waited outside the room during the video recording. To prevent the influence of odors, hair, feces, and urine left by other animals, the OFT was washed with 70% ethanol after each animal was tested. After this procedure, the OFT was left to dry for 15 minutes.

Hole-board test

The hole-board test (HBT) is used to evaluate anxiety-like and exploratory behaviors[26]. Rats examine the holes in the test apparatus and dip their heads through the holes as their anxiety decreases. Dipping their heads through the holes is an exploratory behavior that increases with reduced anxiety. Sixteen holes were used in the experiments. Head-dipping numbers, head-dipping latency, and new-hole-dipping numbers were recorded for 5 minutes. The experimental room was kept quiet under constant lighting (110 Lux, warm light). A camera recorded the test, and the tapes were analyzed blindly. The researchers waited outside the room during the video recording. To prevent the influence of odors, hair, feces, and urine left by other animals, the HBT was washed with 70% ethanol after each animal was tested. After this procedure, the HBT was left to dry for 15 minutes.

Statistical analysis

Statistical analyses were conducted using the GraphPad Prism 10.5 software (Boston, MA, United States). Data were assessed for normality using the Shapiro-Wilk test. Unpaired comparisons showing a normal distribution were evaluated using Student’s t test. Data are presented as the mean ± SD. Data not normally distributed were analyzed using the Mann-Whitney U test. The medians and interquartile ranges are provided. Differences were considered statistically significant at the P < 0.05 level. In addition, the relationships between the behavioral data and blood glucose levels were analyzed using the Pearson correlation test. In accordance with the resource equation method for animal experiments, 6-11 animals are needed for each of the two groups of studies[27]. Therefore, we used seven rats per group.

RESULTS

Blood glucose levels

Blood glucose levels significantly increased 24 hours after STZ application [control (C): 79.71 ± 12.74; DM: 459 ± 46.67; P < 0.0001] (Figure 2A). Similarly, hyperglycemia was observed in the diabetic group on the day of the behavioral experiments (C: 88 ± 13.08; DM: 525.4 ± 65.34; P < 0.0001) (Figure 2B). On the day of sacrifice, glucose levels were significantly higher in the diabetic group than in the control group (C: 83 ± 9.55; DM: 547 ± 52.54; P < 0.0001) (Figure 2C).

Figure 2 Effect of streptozotocin on glucose level. ^dP < 0.0001 vs control group. A: First measurement; B: Blood glucose level on the day of the behavioral experiments; C: Blood glucose during the terminal stage was analyzed. C: Control; DM: Diabetes mellitus.

Body weight

Compared with the control group, DM group did not differ in initial (C: 45.71 ± 6.94; DM: 50.86 ± 3.97) and final (C: 53.14 ± 7.66; DM: 49.57 ± 5.12) body (P > 0.05). The body weight change (C: 7.42 ± 7.87; -1.28 ± 7.5) was lower in the DM group than in the control group (P < 0.05) (Figure 3).

Figure 3 Effect of streptozotocin on body weight. ^aP < 0.05 vs control group. A: Initial body weight B: Final body weight; C: Body weight change. C: Control; DM: Diabetes mellitus.

Behavioral results

According to the OFT behavioral patterns, compared with the control group, the DM group demonstrated a marked decrease in total distance traveled (C: 1057 ± 768.4; DM: 360 ± 296), time spent in the central area (C: 19.86 ± 17.11; DM: 3.71 ± 2.56), and rearing number (C: 7.28 ± 4.27; DM: 2.42 ± 2.76), (P < 0.05; Figure 4). Furthermore, compared with the control group, the DM group displayed significant increases in grooming time (C: 6.71 ± 3.9; DM: 13.57 ± 2.99) and freezing time (C: 11.14 ± 2.61; DM: 32.86 ± 8.23(P < 0.05; Figure 5).

Figure 4 Behaviors in the open-field test. ^aP < 0.05 vs control group. A: Total distance traveled; B: Central zone time C: Rearing number; D: Open field test. C: Control; DM: Diabetes mellitus.

Figure 5 Freezing and grooming behaviors in the open-field test. ^bP < 0.01 vs control group; ^dP < 0.0001 vs control group. A: Freezing time; B: Grooming time. C: Control; DM: Diabetes mellitus.

In the HBT, compared with those in the control group, the number (C: 8.42 ± 1.98; DM: 3.71 ± 2.05; P < 0.001) and duration of new-hole head-dipping episodes (C: 17.14 ± 3.97; DM: 8.14 ± 4.63; P < 0.01) in the diabetes group significantly decreased (Figure 6). Similarly, the total number and duration of head-dipping episodes decreased in the DM group (total head-dipping number, C: 13 ± 5.88; DM: 4.57 ± 3.25; total head-dipping time, C: 25.29 ± 10.98; DM: 9.42 ± 6.75; P < 0.001), (Figure 6). The time to the first head-dipping behavior was prolonged in the DM group (C: 7.14 ± 6.81; median: 4; interquartile range: 14; DM: 62 ± 104.8; median: 25; interquartile range: 40; P < 0.05), (Figure 7).

Figure 6 Behaviors in the hole board test. ^bP < 0.01 vs control group; ^cP < 0.001 vs control group. A: New hole head dipping number; B: New hole head dipping time; C: Total Head dipping number; D: Total head dipping time. C: Control; DM: Diabetes mellitus.

Figure 7 Hole board latency and head dipping behavior in the hole board test. ^aP < 0.05 vs control group. A: Hole board latency; B: Head dipping behavior. C: Control; DM: Diabetes mellitus.

Correlation results

These results are presented in Figure 8. A statistically significant correlation was detected between behavioral patterns (central zone time, rearing number, new-hole head-dipping number, and total head-dipping number) and glucose values (correlations between central zone time and glucose (r = -0.80; P = 0.0289), between rearing and glucose (r = -0.84; P = 0.0174), between new hole head dipping number and glucose (r = -0.91; P = 0.0038), and between total hole head dip count and glucose (r = -0.93; P = 0.0018).

Figure 8 The correlation results. A: The correlation between central zone time and glucose (r = -0.80, P = 0.0289); B: The correlation between rearing and glucose (r = -0.84, P = 0.0174); C: The correlation between new hole head dipping number and glucose (r = -0.91, P = 0.0038); D: The correlation between total hole head dip counts and glucose) (r = -0.93, P = 0.0018).

DISCUSSION

In the present study, diabetes was induced in young rats using STZ. The early-stage effects of diabetes included an increase in anxiety-like behaviors and a decrease in exploratory behavior and locomotor activity. A strong correlation was found between the behavioral data and hyperglycemia.

A dose of 120 mg/kg STZ was selected to establish an STZ- induced diabetes model. According to our results, hyperglycemia consistent with diabetes was observed in 30-day-old rats given a 120 mg/kg dose. Blood glucose levels were measured on three consecutive days after the administration of 120 mg/kg STZ to 30-day-old rats, and all the rats had blood glucose levels above 400 mg/dL. These data were similar to those from other preclinical studies in which 120 mg/kg STZ was administered to juvenile rats[20-24]. The STZ dose varies according to age, sex, and species[28]. Higher doses of STZ are required to induce diabetes in juvenile rats than in adult rats because of continued pancreatic proliferation[28]. STZ is highly toxic to pancreatic beta cells[29]. STZ cannot cross the blood-brain barrier when it is administered intraperitoneally[30]. Therefore, it is indirectly responsible for behavioral changes. Interestingly, when administered intracerebroventricularly at extremely low doses (2-3 mg/kg), STZ, which cannot cross blood-brain barrier, also creates a sporadic Alzheimer’s disease model[31,32].

Although no significant difference in weight was observed between the groups, a marked change in weight loss was observed in the DM group, whereas the control group experienced weight gain. Weight loss after STZ administration is consistent with the findings of other preclinical studies[10,11].

According to our results, anxiety-like behaviors increased in diabetic rats in the OFT. Previous studies conducted on adult rats in the literature have also shown increased anxiety-like behaviors in STZ-induced diabetic models[33-35]. These studies focus on both adult rats and the long-term effects of diabetes on brain health. In the present study, the number of rearing behaviors, a measure of exploratory behavior, decreased significantly as a short-term adverse effect of STZ. Rearing behavior has also been shown to be negatively affected in other rodent studies[36,37]. Furthermore, the locomotor activity of rats was negatively impacted in this study. Other studies measuring locomotor activity in the OFT have reported similar results[38,39]. Rebolledo-Solleiro et al[40] reported that water and food deprivation increased locomotor activity in their STZ studies. The rats exhibited hyperactivity while they were searching for water. Given this factor, regular checks were conducted to ensure that the rats were not deprived of water or food. Furthermore, among the signs indicating increased anxiety in the subjects are grooming and freezing in the OFT[10,11]. Both of these behavioral patterns were observed in diabetic rats in this study. Similarly, grooming and freezing have been reported in studies of adult diabetic rats[10,11].

The HBT is another behavioral test used to analyze anxiety-like behavior[26]. By examining the holes in the experimental apparatus and inserting their heads into them, rats exhibit exploratory behavior[26]. Our data indicate that in early-stage damage, juvenile rats exhibit a significant impairment in the frequency and duration of exploratory head-dipping behavior through the hole. Additionally, the head-dipping latency was adversely prolonged in the diabetes group (Figure 5). This finding is consistent with the results of the OFT, which revealed an increase in anxiety-like behaviors and impaired exploratory behavior in diabetic rats. Other preclinical HBTs and diabetes studies have focused on long-term and adult periods[41,42].

One of the most commonly used tests in anxiety research is the elevated plus maze[43]. Elevated plus maze studies have demonstrated decreases in the time spent in the open arm, the percentage of time spent in the open arm, and the percentage of open-arm entries in rodents[44-46]. In the light-dark box test under high-light conditions, diabetic rats spent more time in the dark, indicating a fear of light[47]. In addition, depression-like behaviors accompanied by decreased swimming time in the forced swim test and increased immobility time in the tail suspension test have been reported[48,49].

According to our results, hyperglycemia is significantly correlated with increased anxiety behaviors and decreased exploratory behaviors. Sommerfield et al[50] reported that hyperglycemia in diabetic patients impaired attention and working memory, leading to sadness and anxiety. Anderson et al[51] reported a relationship between anxiety and poor glycemic control in their meta-analysis. Pan et al[52] reported that hyperglycemia is a significant reason for the development of anxiety through a C-C motif chemokine ligand 2-dependent mechanism. Kwon et al[53] reported that poor glycemic control is associated with psychiatric disorders such as anxiety disorders and depression. Preclinical rodent studies examining the relationship between diabetes and anxiety have reported that, in addition to hyperglycemia, oxidative stress, decreased levels of neurotrophic factors, and increased inflammation are responsible[54-56]. Inflammation and oxidative stress can decrease the production of neurotransmitters and neurotrophic factors in the brain. Furthermore, increased inflammation disrupts the physiological regulation of the hypothalamic-pituitary-adrenal axis[57]. STZ has been reported to disrupt the hypothalamic-pituitary-adrenal axis, decrease neurotrophic factor production, and increase oxidative stress in diabetic animals[58-60]. Studies on STZ-induced diabetes examining anxiety-like behaviors also highlight issues related to these physiological pathways[10,12,56].

This study has certain limitations, the most important of which is that the experiments were conducted only with male rats. This study could have included more animals, in particular, female rats. From the perspective of public health and translational medicine, it would be beneficial to conduct experiments with female rats as well. This limitation was due to budget and time constraints. The behavioral experiments used in this study, combined with other experimental setups, could be conducted with more groups. The number of behavioral tests was limited to two because increasing it would alter behavioral patterns.

CONCLUSION

In the DM model created with STZ, anxiety-like behaviors increased in the early stages of the OFT and the HBT. Exploratory behaviors and locomotor activity decreased dramatically. A strong correlation was found between the increase in anxiety behaviors and the impairment of exploratory behaviors and hyperglycemia. These changes observed in the early stages of DM may be warning signals for families and pediatricians. Further clinical studies involving children and preclinical studies involving juvenile rodents are needed.