Dementia is characterized by a decline in thinking, memory, and independence in daily work. Dementia is a cardinal sign of Alzheimer's disease (AD) causes neurodegeneration due to oxidative stress and neuroinflammation in the brain that are responsible for the production of amyloid beta protein and tau tangles (Yang et al., 2020, Jeremic et al., 2021). The prevalence of Alzheimer's disease increases with age, and it is estimated that about 5–8% of people over the age of 60 have Alzheimer's disease. By the age of 85, the prevalence of Alzheimer's disease is estimated to be around 30–40% (McDade, 2022).
The mouse model of Alzheimer's disease was developed by administering streptozotocin intracerebroventricularly (ICV) with the aid of stereotaxic surgery (Yamini et al., 2022b). STZ induces impairments in various behavioral aspects such as leaning, memory, and cognitive functions through the interference with cerebral insulin signaling, disruption of energy metabolism, and mechanisms including increased neuroinflammation, imbalances in the cholinergic system, oxidative stress in the brain, and dysfunction of existing neurons (Grieb, 2016). STZ was administered twice, with a dose of 1.5 mg/kg per day given after the surgery and a separate dose of 3 mg/kg given on a different day (Nakhate et al., 2018).
Most current therapies for Alzheimer's disease and other CNS disorders utilize peripheral routes of drug administration, such as parenteral and oral administration. However, these approaches often result in diminished efficacy and potency of the medication treatment. Nose-to-brain drug delivery, on the other hand, offers a promising solution by enabling the direct transport of therapeutic molecules without the need to pass through the blood-brain barrier (BBB). This approach significantly enhances the drug concentration in the brain, thereby improving the effectiveness of treatment (Lochhead et al., 2015). Generally, the nasal cavity is an ideal route for drug administration due to its high permeability and effective absorption of both small molecule medications and biopharmaceuticals through the nasal mucosa. Additionally, the proximity of the nasal cavity's roof to the brain, which houses nerves projecting to the brain, further enhances the potential for drug delivery. By bypassing the blood-brain barrier and blood cerebrospinal fluid barrier, nasal-to-brain drug administration offers a promising approach. The delivery of drugs from the nose to the brain is minimally invasive, exhibits good patient compliance, and provides opportunities for self-medication. It is noteworthy that nasal-to-brain delivery is not limited to small molecule medications alone; studies have shown that peptides, proteins, stem cells, viruses, and nucleotides can also traverse from the nose to the brain. Overall, nasal-to-brain drug delivery holds great potential as a non-invasive and effective means of delivering a wide range of therapeutic agents to the brain. (Gänger and Schindowski, 2018).
In the current study, a PLGA-6HOF intra nasal nanoparticle formulation was developed with the objectives of bypassing the blood-brain barrier (BBB). The doses given to the animals were adjusted based on their weight, and the nose-to-brain dosing was conducted using a micropipette with a volume of 5µl, delivered into one nostril(Hong et al., 2019).
In this study we administered freshly prepared intranasal formulation in PLGA for 21 days to experimental animals. The characterization of the formulation was carried out by zeta sizer and we observed that sharp peak, indicating a well-defined particle size distribution of 164.1 nm (Fig. 2), and zeta potential of -18.2 mV (Fig. 3) which lies ins good category of formulation parameter reported in previous studies. (Weng et al., 2020). Also a good %CDR was observed at 60 min (2.26%) and at 300 min (5.5% ) (Fig. 4).
The short-term, long-term, and spatial memory processing that was impacted by Alzheimer's disease (AD) will be evaluated by using The Morris Water Maze, Elevated Plus Maze, Novel Object Recognition, and Y-maze, which are four well-known hippocampus-dependent spatial memory tasks that have been used in the past to evaluate cognitive function (Gamberini et al., 2015, Kraeuter et al., 2019, Tian et al., 2019).
The findings of our study provide compelling evidence that the administration of 6-Hydroxyflavone through an intranasal formulation effectively promotes the development of spatial memory in a mouse model with Alzheimer's disease induced by STZ.
Mice were put through memory tests in the EPM, a paradigm for exteroceptive behavior (Gamberini et al., 2015). Similar changes were seen in our study, where drug therapy reduced the elevated inflexion ratio in the treatment groups. The inflexion ratio (Transfer latency) in the EPM was increased in STZ treated mice. When an intranasal formulation of 6-Hydroxyflavone was used the higher inflexion ratio in comparison to mice treated with STZ made this improvement clear, as seen in Fig. 5. These results imply that the intranasal formulation of 6-Hydroxyflavone (1mg/kg) used in this investigation had a more favorable effect than oral treatment of 6-Hydroxyflavone on the short-term memory inflexion ratio of STZ-treated mice within the EPM. According to Retinasamy et al., 2020 (Retinasamy et al., 2020), the inflexion ratio decreased in the STZ group as compared to the control group. However, treatment caused the inflexion ratio to rise, showing that the disease had improved.
In the Y-Maze, which is widely used to assess spatial learning and memory, spontaneous alternation was employed as a measure of working memory to evaluate the exploratory behavior of mice (Kraeuter et al., 2019). The administration of an intranasal formulation of 6-Hydroxyflavone (1mg/kg) to mice resulted in a significant enhancement of spatial working memory as seen in Fig. 6. This improvement was observed through an increased percentage of spontaneous alternation compared to STZ-treated mice. These findings indicate that the use of 6-Hydroxyflavone in this study exhibited a beneficial effect on short-term memory in STZ-treated mice within the Y-Maze.
To measure recognition memory, novel object recognition was one of a crucial neurobehavioral metrics that takes advantage of mice's innate propensity to look at unfamiliar items rather than familiar ones. The novelty preference reveals how much the mouse prefer the novel object, whereas the discrimination index reveals how much the mouse preferred the familiar object over the novel one (Pi et al., 2020). When mice were administered an intranasal formulation of 6-Hydroxyflavone, they exhibited a significantly longer exploration time of the novel object during the test phase. This increased exploration time suggests a higher preference for novelty and indicates an improvement in cognitive function. Additionally, both the intranasal formulation of 6-Hydroxyflavone and donepezil-treated mice demonstrated a higher discrimination index compared to the STZ-treated group as seen in Fig. 7. This indicates their ability to distinguish between the familiar and novel objects (Sefati et al., (2023) and Akhtar et al (2021),previously reported in their study that after STZ induction, the discrimination index and inflexion ratio significantly decreased.
The Morris water maze test shows learning ability by assessing the latency to reach the platform during training. When the platform was taken down on the fifth day, the period of time spent there, the latency to the target quadrant, and the quantity of platform crossings were all examined (Ma et al., 2014). As shown in Fig. 8 we observed that intranasal formulation of 6-Hydroxyflavone sustain the long-term memory, as 6HOF-NTB and 6HOF p.o treated mice showed decreased in latency to target quadrant (Fig. 8a) and increase in number of platforms crossing(Fig. 8b)and time spent in target quadrant(Fig. 8c) as compared to STZ treated mice.
The cholinergic system plays a vital role in memory and cognition, and its deterioration is one of the major hallmarks of AD. Thus, treatments acting as AChE inhibitors may confer improvement in cholinergic function and hence cause memory restoration(Pohanka et al., 2011, Worek et al., 2012).
Most of the drugs used to treat Alzheimer's patients are acetylcholinesterase inhibitors, which have been demonstrated to improve cognition and hence act as symptomatic treatment. Hence, measuring AChE activity becomes a crucial parameter in proving that our treatment can also work as planned. The acetylcholinesterase assay was performed using the modified Ellman's method (Worek et al., 2012).
Figure 9 showed that administration of STZ significantly increased Acetylcholinesterase levels in brain. However, this increased AChE levels were significantly reduced by the administration of intranasal formulation of 6-Hydroxyflavone. These findings suggest that 6-Hydroxyflavone has the potential to modulate AChE activity, which may have implications for the treatment of AD.
Oxidative stress is a state of imbalance between the production of reactive oxygen species (ROS) and the ability of cells to neutralize or repair the damage caused by ROS. Neurodegeneration refers to the progressive loss of structure or function of neurons in the brain, which can lead to Alzheimer's disease(DeTure and Dickson, 2019). While due to STZ administration increase in oxidative stress was seen in the current study (Bathina et al., 2017).
This Study showed that there was a notable rise in oxidative stress levels in STZ treated mice which assessed by measuring the activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione (GSH), and catalase. Additionally, the levels of malondialdehyde, a marker of lipid peroxidation and oxidative damage were measured (Sodhi and Singh, 2013). The results in Fig. 10 demonstrated that after administration of 6HOF -NTB, significant increase in the activity of antioxidant enzymes, including CAT (Fig. 10a), SOD (Fig. 10b), and GSH (Fig. 10c), indicating an enhanced antioxidant defense system, moreover, there was a decrease in the levels of malondialdehyde(MDA) (Fig. 10d), suggesting reduction in oxidative damage in the brain.
Furthermore, the administration of 6-Hydroxyflavone demonstrated not only a reduction in brain histopathological alterations induced by intracerebroventricular STZ treatment but also a decrease in neutrophil infiltration and the presence of pyknotic cells in brain tissue slices stained with hematoxylin and eosin (H&E) (Fig. 11). These beneficial effects were comparable to those observed with the administration of donepezil.