Development of an Experimental Animal Screening Model for... : Medical Journal of Dr. D.Y. Patil University (2024)

INTRODUCTION

Type-3 diabetes mellitus (T3DM) is a condition that occurs when the brain is unable to utilize or produce insulin. It has been linked to Alzheimer’s disease (AD) and other neurological disorders.^[1] It is known that acetylcholine (ACh) can stimulate the release of insulin from the pancreas, and that insulin can modulate the release of ACh from the brain.^[1] Insulin resistance has been linked to the development of AD, although the exact relationship between the two is still not fully understood. Insulin resistance is associated with an increased risk of AD, as well as other forms of dementia.^[2] It is reported that insulin resistance may lead to increased levels of amyloid-β plaques, which can then lead to the development of AD.^[3] Brain insulin signaling is one of the major factors that contributes toward the pathology of AD. A cohort study conducted on diabetic patients aging over 75 years reported a significant association of uncontrolled T2DM with AD.^[4] Similarly, another study conducted on 824 patients reported 65% increase in the risk of development of AD in the presence of diabetic condition.^[5]

Streptozotocin (STZ) is a chemotherapeutic agent commonly used for the induction of diabetes in experimental animals. STZ can produce insulin resistance by permanently damaging the beta-cells of the pancreas.^[6] Long-term high-fat diet (HFD) can cause cells to become resistant to insulin, which can lead to higher levels of glucose in the blood and an increased risk of developing type-2 diabetes. Insulin resistance may also be related to inflammation and oxidative stress, both of which are known to contribute to Alzheimer’s disease.^[7]

There are several experimental methods are available to induce type-2 diabetes mellitus (T2DM) in experimental animals. Although, no definite method is established to induce T3DM which is associated with T2DM and AD. Here we are presenting the data that may justify the association of high-dose STZ along with HFD and NA for induction of T3DM. Based on our study, we observed that STZ and HFD induced diabetes mellitus animal showed Alzheimer like symptoms. High levels of blood sugar can contribute to oxidative damage and inflammation in the brain, which can lead to the formation of amyloid plaques and neurofibrillary tangles, the hallmark features of Alzheimer’s disease.

MATERIALS AND METHODS

Materials and equipment

Chemicals including STZ [18883-66-4], nicotinamide [98-92-0], ketamine [6740-88-1], and xylazine [7361-61-7] were obtained from Sisco Research Laboratories. Tris-HCl, sodium chloride, and ethylene diamine tetra chloride (EDTA) [60-00-4] were procured from Sigma Aldrich. ACh assay kit and AChE assay kit were collected from Biolabs and AneuRO, respectively. Dounce hom*ogenizer was procured for the preparation of brain hom*ogenate. Digital animal weighing scale (4000 g capacity) was procured from Kent scientific. High-fat diet (HFD) containing food for animal was prepared according to the standard procedure^[8] as given in Table 1.

Animals

Twelve female Albino Wistar rats weighing 250.7 ± 4.4 g were approved by Institute Animal Ethics Committee (IAEC) under IAEC number RKCP/COI/Re22/125 and obtained from animal house of the School of Pharmacy, R.K. University. At an initial stage, animals have been provided with regular food, free access to water, and ideal environmental conditions. Animals were kept in cages at 24.2°C temperature and 30–70% relative humidity. The care of experimental animals was taken as per the committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines.

Induction of Type-3 diabetes

On week 0, animals of a disease control group were fed with a high-fat diet for four weeks to induce insulin resistance. After four weeks, animals were kept for overnight fasting and administered with 230 mg/kg nicotinamide (0.9% saline solution) before the administration of STZ. After 15 min of nicotinamide administration, the animals were injected with 65 mg/kg STZ intraperitoneally prepared in 0.1 M citrate buffer. Furtherly, animals were provided with 10% glucose solution orally for 24 h.^[8] Animals were observed for seven days for mortality.

Estimation of body weight and blood glucose levels

Body weight (BW) of experimental animals was determined throughout the study period of five weeks using an animal weighing scale (Kents scientific). On week 1 and week 4, blood was drawn from the animals using a retro-orbital plexus and stored in fluoride vials. The Trinder’s methodology was used to estimate the blood glucose levels (BGL).^[9]

Estimation of glycosylated hemoglobin levels

On week 5, the glycosylated hemoglobin levels were estimated. Firstly, whole blood was mixed with a lysing reagent consisting of detergent and highly concentrated borate ions. The labile Schiff’s base was removed, and the absorbance of the glycosylated hemoglobin (HbA1c) levels was determined at 415 nm.^[9]

Estimation of behaviors parameters

Morris water maze test

The Morris water maze test was conducted to evaluate spatial learning and memory in rats.^[10] The test involved a circular pool filled with opaque water, a hidden platform, and a training phase consisting of four trials per day for four consecutive days. On the fifth day, the probe trial was conducted to assess the rat’s memory retention. The data were analyzed using a video tracking system, and escape latency (EL) was measured.

T maze model

Animals were trained to get familiar with the maze environment and to locate the goal box correctly.^[11] This training was done for two days before the test day. On the test day, each rat was placed in the start box, and the door was lifted to allow the rat to explore the maze. The rat had to choose between two goal boxes at the end of each arm. The test consisted of ten trials with a 15-min interval between each trial to avoid memory bias. The time taken by the rat to reach the correct goal box was recorded for each trial and recorded as the elapsed time in seconds. The data obtained from this test were analyzed using appropriate statistical methods to determine the cognitive performance of the rats.

Rota-rod apparatus

Animals were assessed for their motor coordination and balance using a standard procedure.^[12] The apparatus used was a rotating rod with a diameter of 7 cm, divided into four compartments. The speed of the rod was set at 16 rpm for the initial trial and gradually increased to 40 rpm over the course of five minutes. Before testing, rats were trained for three consecutive days at a constant speed of 10 rpm for five minutes. On the test day, each rat was placed on the rod and allowed to walk until it fell off or reached the maximum time of 300 seconds. Each rat was tested three times with a 15-min interval between each trial. The latency to fall from the rod was recorded for each trial, and the average latency was calculated for each rat.

Preparation of brain hom*ogenate

The animals were euthanized using the overdose of ketamine and xylazine. The preparation of brain hom*ogenate was carried out according to a standard protocol.^[13] Fresh brain tissues were collected and immediately placed on ice to prevent degradation. The brain tissue of three animals of the group was then hom*ogenized using a Dounce hom*ogenizer in ice-cold hom*ogenization buffer containing Tris-HCl, NaCl, and EDTA, at pH 7.4. The hom*ogenate was centrifuged at 10,000 rpm for 15 min at 4°C to separate the crude membrane fraction and the supernatant. The supernatant was then transferred to a fresh tube and centrifuged again at 14,000 rpm for 30 min at 4°C to obtain the final hom*ogenate.

Estimation of acetylcholine and acetylcholinesterase

Levels of ACh and AChE in the brain hom*ogenate were estimated using commercialized Cell Biolabs’ Acetylcholine Assay kit and AneuRO Acetylcholinesterase Assay kit, respectively. The procedure was undertaken as per the manufacturer’s instructions.

Histopathological analysis

The hippocampus region of brain tissues of three animals of each group was fixed in 10% buffered formalin for 24 h and then processed for paraffin embedding. The tissue was dehydrated in graded alcohol series, cleared in xylene, and embedded in paraffin wax. The paraffin-embedded tissue was then sectioned at 3 microns thickness using a rotary microtome and mounted on glass slides. The slides were then deparaffinized in xylene, rehydrated in graded alcohol series, and stained with hematoxylin and eosin stain. The stained slides were examined under a light microscope. The histopathological changes were evaluated and recorded.

Statistical analysis

Data were presented in the form of mean ± standard error of the mean (SEM). The statistical analysis was performed using a two-way analysis of variance (ANOVA). P value < 0.05 was considered a statistically significant measure. Results were analyzed using GraphPad Prism 8.0.2.

RESULTS

Body weight and blood glucose levels

No significant changes in BW and BGL were observed in the treatment period of week 1 and week 2. Animals of disease control group showed significant increase in BW and BGL on week 3 and week 4 of the induction period with P value < 0.05. Figure 1a and Figure 1b showcases graphical presentation of changes in BW and BGL, respectively.

Glycosylated hemoglobin levels

On week 4, animals of disease group showed significant elevation in the levels of HbA1C compared to the normal control group with P value < 0.0001. Figure 2 shows a graphical representation of changes in glycosylated HbA1C level.

Behavioral parameters

Morris water maze test

Animals showed no difference in EL on week 0. Over the treatment period of four weeks, disease control group showed increase in EL. In the baseline, the increase in escape latency was not significant. In week 1, we observed statistically significant increase in EL with P value < 0.05. In week 2, week 3, and week 4, the statistically significant elevation was observed in the disease group with P value < 0.001 (Figure 3a).

T maze model

Animals showed no difference in the elapsed time on week 0. On week 1, a significant increase in the elapsed time was observed in disease group compared with normal group with P value < 0.05 [Figure 3b]. The difference of elapsed time between disease group and normal group increased significantly during week 2, week 3, and week 4 with P value < 0.001.

Rota-rod apparatus

Animals showed no difference in the elapsed time on week 0. On week 1, elapsed time decreased significantly in disease group with P value < 0.05 [Figure 3c]. Similarly, animals of disease group showed a significant reduction (P value < 0.001) in elapsed time in week 2, week 3, and week 4.

Levels of Acetylcholine and Acetylcholinesterase

Animals of disease control group showed significant reduction in the levels of ACh in comparison with the normal control group over the treatment period of four weeks with P value < 0.0001 [Figure 4a]. Levels of AChE increased significantly in disease control group over the treatment period of four weeks with P value < 0.0001 [Figure 4b].

HISTOPATHOLOGY

In histopathological analysis, animals of normal control group elucidated normal morphology of hippocampus cells at 40x magnification [Figure 5a]. Small number of β-amyloid plague was observed in animals of normal control at 100x magnification [Figure 5b]. Animals of the disease control group showed irregular morphology of hippocampus cells at 40x magnification [Figure 5c]. A notable increased number of β-amyloid plaque were visible in animals of disease control at 100x magnification [Figure 5d].

DISCUSSION

Type-3 diabetes mellitus refers to the circ*mstantial presence of type-2 diabetes along with cognitive impairment resembling the symptoms of AD. A study confirmed that the development of insulin resistance may lead to cognitive decline and AD.^[14] Multiple studies have found that individuals with diabetes have a higher risk of developing AD and that the two conditions share common features including insulin resistance and impair glucose metabolism and inflammation.^[15-17] The mechanisms underlying in brain insulin resistance in this condition include impaired insulin signaling, inflammation, oxidative stress, and mitochondrial dysfunction.^[18] The brain relies on glucose for energy, and insulin resistance may impair glucose uptake and metabolism in the brain leading to neuronal damage and cognitive decline. A study demonstrates that high sugar levels can cause damage to blood vessels in the brain leading to reduced blood flow and oxygen delivery to brain cells that leads to AD.^[19] Insulin resistance in the brain may lead to the accumulation of β-amyloid, a protein that forms the plagues that are a hallmark of AD.^[20] Managing diabetes and controlling blood sugar levels may be an important strategy for reducing the risk of AD.

Streptozotocin is preliminarily used to induce diabetes mellitus in experimental animals by irreversibly damaging insulin-producing β-cells in the pancreas and thus causing insulin resistance.^[21] A study has confirmed that when 3 mg/kg STZ was injected through an intracerebroventricular (ICV) route into the brain of rats, it caused memory impairment and oxidative stress which are the features of AD.^[22] The exact mechanism by which ICV injection of STZ causes memory impairment is still not fully understood. However, it is believed that STZ induces oxidative stress, inflammation, and insulin resistance in the brain, which can lead to the accumulation of amyloid beta and tau hyperphosphorylation.^[22] These changes can ultimately result in cognitive impairment and other AD-related symptoms. A study established that administration of HFD in experimental animals resulted in the excessive accumulation of saturated fatty acids and simple sugars inflammation and oxidative stress in the body that may interfere with insulin signaling pathways and contribute to insulin resistance.^[23] Additionally, high-fat diets can lead to increased accumulation of lipids in non-adipose tissues such as the liver and skeletal muscle, which can also impair insulin sensitivity.^[23]

We postulated that administration of HFD for four weeks followed by the high dose (65 mg/kg) of STZ and nicotinamide (230 mg/kg) given intraperitoneally may result in the induction of T3DM. Nicotinamide was administered 15 min before the STZ injection to reduce mortality in experimental animals. Obesity is one of the factors that contributes in the pathophysiology of T2D and AD. We observed a significant increase in the body weight suggesting the presence of obesity. Secondly, BGL is the preliminary factor affecting T2DM and insulin levels. Elevated levels of BGL may confirm the presence of T2DM. Findings of the study also suggest the increased levels of HbA1c which is found to be associated with AD.^[24] Behavior parameters conducted in the study confirm the cognitive impairment in the experimental animals. We observed a significant reduction in the animals’ cognitive abilities through the behavior parameters. Reduction in Ach levels can be observed due to the degeneration of cholinergic neurons, which are responsible for producing and releasing ACh. ACh is involved in cognitive functions such as learning, memory, and attention, and its deficiency contributes to the cognitive decline seen in AD. However, in AD, there is an increase in the activity of the enzyme acetylcholinesterase (AChE), which breaks down ACh in the synaptic cleft. This increased activity of AChE further exacerbates the deficiency of ACh in the brain. Results of study showed decreased levels of Ach and increased levels of AChE confirming the reduction in cholinergic neurons and ultimately resembling AD condition. From the histopathological analysis, we observed a notable increase in the number of β-amyloid plagues and inflammation which are also suggesting the presence of AD.

The study emphasizes the probable induction of T3DM by the intraperitoneal route in experimental animals. Although, the molecular mechanism behind this action is still unclear. We did not observe any mortalities during the study period. Therefore, the proposed method can be relatively termed as simpler, effective, and safer to induce T3DM.

CONCLUSION

The results of the study imply the induction of T3DM through intraperitoneal administration of STZ and nicotinamide in animals fed with HFD for four weeks. Animals of DC group showed statistically significant changes in parameters related to diabetes and dementia in comparison with the NC group. The presence of diabetic symptoms and dementia probably suggests T3DM condition. Further investigations can be done evaluating the molecular mechanism behind the progression of the disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

REFERENCES