Diabetes is a chronic disorder, which is relatively common throughout the world (Eraslan, 2026). Diabetes is associated with alteration of functional and anatomical structure of various organs of the body (Khajuria, 2018). The long-term complication of diabetes affects microvascular that further may also leads to neuropathy. Streptozotocin (STZ) is a chemical derived from Streptomyces achromogenes, which is used to induced type I diabetes in rat model with single dose. It spontaneously produces nitric oxide, which is identified as a potent pancreatic islet toxic compound and responsible for the necrosis of pancreatic beta cells (Rais, 2022). Nitric oxide activates metabolic or signaling pathways and generates more reactive oxygen species (ROS) leading to oxidative stress and failure of beta cell of pancreas. Body temperature is affected by over production of nitric oxide, it acts as thermoregulator. In STZ induced diabetic condition, the body weight reduced due to muscles breakdown as well as dehydration after frequent urination in subject with high blood glucose level (Gocmez, 2019). On the other hand, Lipopolysaccharide (LPS) was employed as an established neuroinflammatory Alzheimer’s-like model to validate whether progressive diabetes produces comparable behavioral and histological features of neurodegeneration. Similarities between both models would support a mechanistic link between chronic diabetes and Alzheimer’s disease progression.
       
The review of literature reveals that in long term diabetic and hyperglycemic conditions, memory loss is evident due to damage in neurons of limbic system such as amygdala, thalamus, hypothalamus, neural network, region of hippocampal and nerves of cerebrum of brain (Contreras, 2017). Although both STZ induced diabetes and LPS induced neuroinflammation are widely used experimental models to study cognitive impairment and neurodegeneration, limited studies have directly compared these two models under similar experimental conditions. Diabetes primarily reflects chronic metabolic dysfunction, hyperglycemia, insulin resistance and oxidative stress, whereas Alzheimer’s disease (AD)-like pathology represents acute neuroinflammation-mediated cognitive decline (Mandour, 2021). However, the relative contribution of these distinct pathological mechanisms to behavioral deficits and brain histological alterations remains unclear. In particular, there is insufficient evidence correlating behavioral impairments with histopathological changes in key brain regions such as the hippocampus and cortex between these two models. A comparative evaluation is therefore needed to determine whether metabolic injury or inflammatory insult produces more severe neurobehavioral and neuronal damage.
       
We hypothesize that both diabetic and AD rats will exhibit significant behavioral impairments and histological abnormalities compared with control animals. However, the long- term diabetes model is expected to produce progressive cognitive and motor deficits associated with neuronal degeneration through chronic metabolic stress, while the LPS-induced AD model is anticipated to show more pronounced memory impairment and neuroinflammatory histopathological changes. Furthermore, we hypothesize that the severity of behavioral dysfunction will positively correlate with the extent of neuronal loss, gliosis and tissue disorganization in the hippocampus and cerebral cortex.

Subjects
 
Male Wister rats of 10-12 weeks of age (n=24), weight 185±15 gm were obtained from the animal house of Birla Institute of Technology, Mesra, Ranchi (India). These rats were equally divided into three groups: (i) Control (ii) Diabetic and (iii) Alzheimer’s. The animals were acclimatized under the constant 12 h:12 h dark- light cycle in a temperature-controlled room (23±2°C) with food and water ad libitum and housed in individual cages before the experiments. The experimental protocol has been approved by the Institutional Animal Ethics Committee (IAEC) of the Birla Institute of Technology Mesra, Ranchi, Jharkhand, India and granted the ethical approval for this study (1972/PH/BIT/07/23/IAEC). The experiments were performed following the ethical guidelines of IAEC.
 
Experimental rat models
 
Group I (control group; n=8)
 
Control group was injected with 0.5±0.1ml normal saline intraperitoneally in place of drug on the starting day of the experiment.
 
Group II (Diabetic group; n=8)
 
Diabetes in the rats was induced by a single intraperitoneal injection of STZ (65mg/Kg body weight obtained from Sigma-Aldrich, USA) freshly diluted in citrate buffer 0.1 M Na citrate; pH 4.5 to maintain the stability of STZ (Furman, 2021).

Group III (AD group; n=8)
 
Alzheimer’s disease (AD) was induced in a group of rats by a single intraperitoneal injection of lipopolysaccharides (LPS) (10 mg/Kg body weight obtained from sigma-aldrich, USA) freshly diluted in phosphate buffered saline (PBS) (Zhong, 2018).
 
Experimental design
 
In this study, body temperature was measured with the help of a digital telethermometer (Micron digital telethermometer, India) and thermistor probe. Body weight was measured with the help of a common animal weighing machine (Docbel braun company, India, 2 kg capacity). Blood droplet taken from the animal’s tail was used to determine the blood glucose level of the rats by clinical Glucometer (Accu-Chek Instant S brand, Germany) (Qinna, 2015). Body weight, body temperature and blood glucose level were measured before the injection of STZ and after 30 minutes of STZ injection (considered the start date of experiment) followed by every day for one week and latter continued every week till 4 months. Behavioral test in Y maze tools conducted in the same experimental protocol but in LPS treated rats, all the protocol of experiment was followed for the control rats while injecting the 0.5±0.1 ml normal saline in place of STZ and LPS on the starting day of experiment.
 
Recording of behavioral parameters
 
On Y maze, two cognitive tests were performed: (a) Recognition and (b) Spatial spontaneous test.
 
Recognition memory (RM) test
 
To analyze the neurological and cognitive impairments, it is important to test learning and memory in rats. For this test, the subjects were placed on the Y maze for 15 minutes. The Y maze tool apparatus has two equal arms and one different arm used to assess short term spatial working memory and functioning of prefrontal cortex region of the brain and the recognition learning and memory is based on the hippocampus region inter-trial-interval (Popoviç, 2001).  The Y maze recognition test assists more time in novel (closed) arm on second trial. In this test, one arm of Y maze is closed (novel arm) in the first trial and after certain inter-trial-interval (ITI) the novel arm is then opened and then exploratory behavior of rat in all three arms was calculated. The recognition memory is directly correlated with the time in novel arm on the second trial. The calculation of time spent in novel arm has been done with the following formula:

 
Spatial spontaneous alteration (SSA) test
 
Spontaneous alternation test using Y maze is meant to assess short term spatial working memory and hence functioning of prefrontal cortex region of the brain. A spontaneous alternation occurs when a rat enters 3 different arms of the maze in each consecutive arm entry (Miedel, 2017).  It assumes that rats will remember the arms previously visited and show a tendency to enter less visited arms. Firstly, acclimatization or habituation of subjects to laboratory conditions and experimenters for 2 days or up to 1 week. To perform this test, rats are placed at the center of Y maze for 15-minute duration per subject. After total entry in all three arms count the spontaneous alternation and calculate the percentage of spontaneous alteration and reduction in these shows that there is loss in memory and cognition impairment occurs (Ghafouri, 2016).
 
Histopathological observation
 
In case of STZ treatment (diabetic group) after 4 months of experiments and 93 days after LPS treatment animals (Alzheimer’s disease group) were sacrificed quickly, dissected, sample of the brain, liver and pancreas were removed and wash with phosphate buffer saline (PBS), which is an isotonic solution and prevent the rupture of the cells, after that it was fixed in 10% formalin for 24 hours. Before making the slides firstly, preparation of block with paraffin wax (Agrawal Drug Pvt. Limited ME-6681, India) and selected tissue in which wants to investigate about changes in brain, liver and pancreatic cells. After block preparation cut the section at 3.5ì by using Semi Automated Microtome i SAM-17 (Infinity, India, serial no. 2022-23/573) (Samarghandian, 2016). The tissue sections were mounted on glass slides using hot water bath (Yowa water bath, India,220/230 Volt/1/CY.AC.). Afterward, sections of tissue were deparaffinized by xylene and rehydration process by different graded ethanol solution (100%, 90% and 60%). The tissue sections were stained with Hematoxylin and Eosin (HandE) Further all slides were examined using light microscopy [Leica Microsystems Wetzlar, Germany, QmbH, K3C (11547114)] under a magnification of ×100.
 
Statistical analysis
 
The data were statistically analyzed by using origin pro (Origin Lab software, Northampton, Massachusetts, USA). The quantitative variables were expressed as mean±SE. The statistical significance of comparative group was analyzed by the one-way ANOVA followed by Tukey’s multiple comparison post hoc test. Differences were considered statically significant when ‘P’ was less than 0.05 and 0.01.

Analysis of changes in marker parameters in STZ treated diabetic rats
 
The single dose STZ treatment in this study was found responsible for significant increase in blood glucose level as well as reduction in body weight and body temperature just after 48 hours and 15 weeks of the drug treatment in comparison to the control subjects. On third day after treatment of STZ, steep rise in blood glucose level in drug treated rats was recorded (482±40 mg/dL) compared to 104±12 mg/dL in controls (P<0.05). After 4 months, blood glucose level in drug treated rats were recorded as 135±12 mg/dL compared to 92±5 mg/dL in controls (P<0.05). The value of these parameters was shown progressive reduction day by day and returned at the level of the control nearly on the 93^rd day of the experiment. The body weight of the subjects shown significant reduction (P<0.01) after 48 hours of the STZ injection that reached near to the level of the control subjects near the 93^rd day of experiment. The initial weight of experimental animals was 165±15 gm that reduced significantly (P<0.01) to 125±10 gm after the 48 hours of STZ induction. It was interesting that after the drug treatment, the body temperature also falls significantly (P<0.01) from 36.8±0.12°C to 34.4±0.7°C, which was being elevated with decreasing blood sugar level and returned to 37±1°C slowly with the progress of experimental day, there were no significant change in body weight, blood glucose level and body temperature in LPS treated animals as compare to the control group (Table 1).


       
Behavior analysis in Y-maze depicts the Mean±SE of spatial spontaneous alteration and recognition test among all tested groups of rats. Recognition memory percentage performance revealed significantly (P<0.001) decreased across disease groups, indicating progressive cognitive dysfunction (Table 2). Similarly, SSA % showed a significant, time-dependent decline in diabetic and AD groups compared to control, with the most pronounced impairment observed in the combined group at later time points (p<0.001) (Table 3) and (Fig 1).






 
Analysis of changes in histology of brain
 
The parasagittal section of control, diabetic and AD groups of rats revealed almost similar histological structure of hippocampal area, cerebral cortes region and lightly or deeply stained of glial cells, pyramidal cells histology of brain was shown in (Fig 2). Microscopic investigation of brain of control rat shows normal appearance of hippocampal region, neurofibrillary tangles, there were not found any stem cell rest but in diabetic and AD groups of parasagittal section of rats’ brain exhibited the apparent disruption of hippocampal area, disarrangement of most of the pyramidal cell bodies, accumulation of stem rest cell, aggregation of extracellular senile plaque and neurofibrillary tangles.


 
Analysis of changes in histology of liver
 
There were various changes observed in microscopic structure of STZ and LPS treated liver such as changes in parenchymal cells, hydropic swollen, granular degradation, blood sinusoids, portal area and accumulation of glycogen as compare to control all parameters are in normal condition (Fig 3).


 
Histological analysis of drug treated pancreas
 
The ultrastructure changes in drug treated animals shown the empty space, which were previously occupied by the islets among the acinar cells, so that in pancreas observed that entirely loss of beta cell, swollen acinar cell with small vacuoles and alteration in cellular morphology and architecture were found in drug treated rat as compare to control (Fig 4).


       
The analyses of data suggest that the STZ induced a high glucose level on the third day of STZ induction that started decreasing from the third week of drug induction and it normalized with time. Results also showed a significant decrease in body weight and body temperature of the subjects in this experiment. Although, the decreased body temperature returned to its normal range, interestingly the body weight first returned to normal then gone up in these experimental days. STZ reported to enter in the pancreatic beta cell via GLUT2 receptor and produced more nitric oxide and it acts as a temperature regulator causes hypoxia induced hypothermia. Overproduction of nitric oxide activates many metabolic or signaling pathways and generate more reactive oxygen species (ROS) leading to oxidative stress and further to the failure of beta cells of pancreases (Gerber, 2017). In this pathophysiological condition, the loss in body weight is evident due to the capacity of adipocytes to oxidize glucose were remarkably reduces and other factors are dehydration owing to frequent urination as well as the muscles breakdown. Further, these altered changes in blood sugar, body temperature and body weight were analyzed to be temporary as they start to become normal with time.
       
The main finding of this study was the investigation of changes brain calls, hepatic cell and pancreatic cells after STZ and LPS treatment in rats. Histological examination of brain of diabetic rat showed a destruction in hippocampal area, neurofibrillary tangles (Kamat, 2015). Hippocampal area is tilted profile in 3-dimension so that it viewed more dorsally in rodents, used in learning and memory, it controls the spatial navigation, emotional behavior and regulation of hypothalamic function on the other hand aggregation of neurofibrillary tangles composed the hyper- phosphorylation and the aggregation of beta amyloid in senile plaques were analysed by using electron microscopy (González, 2022). The ependymal cells form a simple layer of specialized, ciliated, cuboidal epithelium, which covers with surface of the brain ventricles and spinal cord central canal these differentiated cells from germinal cells of the embryonic neural tube, in disease condition undifferentiated stem cells fails to migrate and found as cluster which is stem cell rest. Destruction of hippocampal region, stem cell rest clustered is mainly due to STZ and LPS induction in different groups of animals.
       
Liver is one of the most important organs that maintains blood glucose levels within normal limits. Histological changes in liver cells such as central vein shows dilated with damage of its lining epithelium, portal vein surrounded with inflammatory cells. In normal condition hepatic artery surrounded with connective tissue but in STZ treated rat it surrounded with inflammatory cells. These changes occur due to enhancement of blood sugar yield to imbalance the oxidation-reduction reactions in hepatocytes, so that, hyperglycemia through increasing in AGEs (advanced glycation end products) facilities free radicals’ production via disturbance in ROS (reactive oxygen species) (Vlassara, 2002). Hence, it reveals that, diabetic hepatic injuries result from several agents and is not controllable only via inhibition of hyperglycemia. After a few months enlargement of liver was observed due to enhancement of triglyceride accumulation by increasing influx of fatty acid into liver induced by hyperinsulinemia (Kraegen, 2001).
       
The histology and the ultrastructure demonstrated that most of the islets were affected and showed notable changes in structures. The beta cells showed degranulation and swelling of the intracellular organelles. It has been reported that the morphological studies have demonstrated, both in vivo and in vitro, that B cell death occurs by necrosis and not apoptosis (Petzold, 2015). Ribosomes of the rough endoplasmic reticulum, the golgi apparatus and the secretory granules are actively involved in the synthesis and the secretion of proinsulin and insulin. All these vital intracellular structures were affected thus inhibiting the synthesis and release of insulin. Without the release of insulin, high FPG levels occur. As reported that STZ-affected cells displayed a variety of changes in their nucleus. Most of the nuclear changes were consistent with Necrotic nuclei contain random nonuniform clumping with indistinct borders and often display nuclear membrane breakdown, cell membrane swelling and rupture, swollen mitochondria with flocculent densities and large swollen endoplasmic reticulum (Mythili, 2004).
       
Many previous studies demonstrated the function of STZ in rat models. Previous studies have shown the reduction in body temperature within 48 hours after STZ induction (Rodrigues, 2018). Due to strong connection between circadian and metabolic pathologies, disrupted body temperature has been observed (Refinetti, 2020). Accumulation of high blood glucose and insulin resistance level is the main reason behind the loss of weight as low insulin cannot uptake glucose from blood and other cells cannot receive the glucose. As a result of this, the energy produced from glucose becomes less and metabolic rate will become slow decrease body weight. Various histological changes were demonstrated that exposer of STZ drug on organs of rats after administration.

Present findings of work support the STZ-induced diabetes and LPS-induced AD models both caused marked behavioral deficits and tissue pathology, indicating a close association between metabolic dysfunction and neurodegenerative changes. Histopathological findings provide valuable insight into disease mechanisms and may support the development of novel therapeutic strategies for diabetes-associated cognitive decline and Alzheimer’s disease. This study highlights the clinical importance of early diagnosis and treatment of diabetes.

The authors would like to express their sincere gratitude to Dr. Yogender Aggarwal and colleagues for their invaluable guidance, continuous support, technical assistance, and constructive suggestions throughout the course of this research. The authors also gratefully acknowledge the Institutional Animal Ethics Committee (IAEC), Birla Institute of Technology Mesra, Ranchi, for granting ethical approval to conduct this study and for ensuring compliance with ethical standards for animal experimentation.
 
Disclaimers
 
The views and opinions expressed in this research paper are strictly of the authors and do not necessarily reflect it’s the official policy or its affiliation. analysis performed in this paper are only premises nd are based on the authors independent interpretations.
 
Informed consent
 
All animal procedures protocol has been approved by the Institutional Animal Ethics Committee (IAEC) of the Birla Institute of Technology Mesra, Ranchi, Jharkhand, India and granted the ethical approval for this study (1972/PH/BIT/07/23/IAEC). The experiments were performed following the ethical guidelines of IAEC.

The authors declare that there are no conflicts of interest regarding the publication of this article.