Table 1.

Pre-clinical experimental animal model using neurotoxin streptozotocin ethidium-mediated tau hyperphosphorylation

S. No.	Dose and Route	Stereotaxic Co-ordinates	Key Points	Behavioral Parameters	Biochemical Parameters	Conclusion	Reference
1	ICV-STZ3 mg/kg bilaterally	AP: 0.8 mm ML: 1.5 mm DV: 4.0 mm	•SIRT1 •Tau phosphorylation ERK1/2 •Streptozotocin	Morris water maze test	•Western blotting •NAD/NADH ratio assay •Co-immunoprecipitation •Measurement activity of SIRT1 deacetylase	•Inactivation of SIRT1, tau hyperphosphorylation,and memory impairment occurred in ICV-STZ-treated rats •Activation of SIRT1 by Resveratrol attenuated tau hyperphosphorylation and memory impairment via inhibiting ERK1/2 activity	[238]
2	ICV-STZ 3 mg/kg bilaterally Volume = 10 μl	AP: 0.8 mm ML: 1.5 mm DV: 3.6 mm	•Xanthoceraside •Learning and memory •Hyperphosphorylated tau •PI3K, Akt, protein kinases •Phosphatases	•Y-Maze test •Novel object recognition test	•Western blot •Phosphorylation level of PI3K,GSK-3β,PP2A	•Xanthoceraside has protective effect against learning and memory impairments •Inhibits tau hyperphosphorylationin the hippocampus through the inhibition of the PI3K/Akt-dependent GSK-3β signaling pathway and increases phosphatases activity.	[239]
3.	ICV-STZ 3 mg/kg bilaterally Volume = 0.5 μl/min	AP: –0.5 mm ML: 1.1 mm DV: –2.8 mm	•AD •Streptozotocin •Nox2 •Cytokines	Object recognition test	•Analysis of cytokines like IL-4,IL-1β,IL-2,10,IFN-γ,TNF-α,IL-12/23 •Nox2 mRNA expression evaluated by RT-PCR in hippocampus •Level of Ox-42 protein expression, a microglial cell marker. •Analysis of GFAP, an astrocyte marker •Level of 4-HNE during lipid peroxidation •Level of 3-NT,marker of oxidative damage induced by tyrosine nitration •Expression ofapoptosis-inducing factor	•Nox2-dependent oxidative stress increase. •Nox2 deletion prevented tau phosphorylation, Aβ expression and decreased neuroinflammation in hippocampus	[240]
4.	ICV-STZ 3 mg/kg bilaterally Volume = 10 μl	AP: 0.8 mm ML: 1.5 mm DV: 4.0 mm	•Adiponectin •Glycogen synthase kinase-3β •AD	•Morris water maze test	•Golgi stain •Western blotting of Tau205, Tau396, Tau404 and Tau5, GSK-3β, GSK-3β (Ser9) and GSK-3β (Tyr216), Akt, p-Akt, PI3K and p-PI3K	•Adiponectin supplements attenuate ICV-STZ-induced spatial cognitive deficits and tau hyperphosphorylation •Inactivate GSK-3β by increasingPI3K/Akt activity	[241]
5.	ICV-STZ 3 mg/kg bilaterally in both lateral ventricles	AP: 0.8 mm ML: 1.5 mm DV: 3.6 mm	•ICV-STZ •Oxidative stress •Tau hyperphosphorylation •Tenuigenin	Morris water maze test	•Nissl staining •Activities of SOD, glutathione peroxidase, and MDA in the hippocampus were measured	•Western blotting: Analysis of 4-HNE protein and phosphorylated tau protein. •SOD activity, glutathione peroxidase, and malondialdehyde contents in the hippocampus were estimated.	[242]
6.	ICV-STZ 3 mg/kg bilaterally in both lateral ventricles Volume = 1 μl/ventricle	AP: 0.8 mm ML: 1.6 mm DV: 4.0 mm	•Streptozotocin •Edaravone •Oxidative stress •Tau hyperphosphorylation	•Balance beam test •Morris water maze test	•Total protein assay •Nissl staining •Western blot was •used to assay the levels of 4-HNE protein adducts, tau, ser396- •phosphorylated tau and thr181-phosphorylated tau	•The analysis of T-SOD, MDA,glutathione, H2O2 and OH, andprotein carbonyl levels were performed	[243]
7.	ICV-STZ3 mg/kg bilaterally Volume = 10 μl on each site	AP: 0.8 mm ML: 1.5 mm DV: 4.0 mm	•PI3K/Akt pathway •GSK-3β •Magnesium sulphate •Tau hyperphosphorylation	•Morris water maze test	•Long term potentiation •Atomic absorption spectroscopy •Golgi staining •Immunohistochemistry •Real-Time Quantitative PCR •Western blotting •Protein expression of IR •Expression of postsynaptic PSD95, PSD93, GLUR1,GLUR2 •Level of GSK-3β,Akt •Expression of synapsin	•GSK-3β and PP2A regulate tau phosphorylation. •ICV-STZ induces inactivation of PI3K/Akt signaling pathway and activation of GSK-3β and cause insulin desensitization. •Magnesium could promote the protein expression of IR,the mRNA levels of insulin and IRimproved synaptic efficacy, and prevented memory and learning impairments	[244]
8.	ICV-STZ 3 mg/kg bilaterally	AP: 0.8 mm ML: 1.5 mm DV: 4.0 mm	•AD •AMPK •Diabetes mellitus Mitochondria tau	•Morris water maze test	•Mitochondrial membrane potential, complex I •activity, and ATP levels assays •Western blotting- p-AMPK,Tau 5 and α-tubulin •Measurement activity of SIRT1 deacetylase •ROS measurement •SOD assay •Estimation of SOD, ROS, Mitochondrial membrane potential, complex I activity, and ATP levels assays	•AMPK isa serine/threonine protein kinase maintainscellular energy balance in mammalian cells. •Reduction in AMPK inducestau hyperphosphorylation in ICV-STZ rats. •Restoring AMPK with its specific activator AICAR can attenuate mitochondria dysfunction, redox dysregulation, cleaved caspase-3, tau hyperphosphorylation, and cognitive impairment.	[245]
9.	ICV-STZ 3 mg/kg injected bilaterally Volume = 1.5 μl was injected in each hemisphere	AP: –0.5 mm ML: 1.1 mm DV: –2.8 mm	•AD •Aβ •Neurofilaments •Streptozotocin •Synapsin •Tau protein	Novel Object Recognition test	•Immunoblotting •Level of ChAT protein •Level of synapsin •Level of NF-L in hippocampus •Estimation of GSK-3β, tyrosineregulated kinase 1A, P25/cdk5, and MAPK, PP1, PP2A, and PP5	•Synapsin is a phosphoprotein related to synaptic vesicles. •Increase activity protein kinases such as glycogen synthase kinase 3β, dual-specific tyrosine regulated kinase 1A, P25/cdk5, MAPK have been observed in AD brains •Decrease in activity of phosphatases such as PP1, PP2A, and PP5, Aβ and increasephos phorylation of Tau, and decreased synapsin expression levels at 14 days after icv injection of STZ	[246]
10.	ICV-STZ 3 mg/kg bilaterally in both lateral ventricles Volume = 5 μl on each site	AP: 0.8 mm ML: 1.5 mm DV: 3.8 mm	•AD •Naringenin •Aβ •Tau •PPARγ	•Morris water maze test •Place navigation test •Body weight •Brain weight	•Glucose measurements •Western-blot analysis- assess the levels of Tau, phospho-Tau, total GSK-3β, phospho-GSK-3β (ser9), IDE and β-actin •Immunohistochemical staining studies •Real-time quantitative RT-PCR=PPARγ and IDE were measured •Expression of AChE •Activity of GSK-3β	•The balance between kinase and phosphatase activities determines the level of p-Tau. •GSK-3 is the S/T kinase that has two isozymes, GSK-3β and GSK-3α. •The GSK-3α regulates amyloidogenic processing of AβPP and GSK-3βkinase in PI3 K/AKT/GSK-3 signaling is regulated by insulin. GSK-3β regulates tau phosphorylation •Increased GSK-3β activity could promote the hyperphosphorylation of tau via inhibiting insulin signaling.	[247]
11.	ICV-STZ 3 mg/kg in one lateral ventricle, i.e., unilaterally Volume = 10 μl	AP: 0.8 mm ML: 1.5 mm DV: 3.6 mm	•AD •Geniposide •Glucagon-like •peptide-1 receptor •Glycogen synthase kinase-3β •Tau hyperphosphorylation •Type 2 diabetes	•Morris water maze test	•Immunohistochemistry •Western blot Assay •Observe PHF using TEM •Estimation of GSK-3β	•GSK-3β is key regulator in glycogen synthesis. •Phosphorylation at tyrosine-216 in GSK-3β or tyrosine-279 in GSK-3α enhances the enzymatic activity, while phosphorylation of serine-9 in GSK-3β or serine-21 in GSK-3α decreases the activity. •Geniposide may serve as a GSK-3 inhibitor to exert its neuroprotective effect in the AD animal model.	[248]
12.	ICV-STZ 3 mg/kg unilaterally in left ventricle only Volume = 3 μl	AP: –1.0 mm ML: 0.3 mm DV: –2.5 mm	•Streptozotocin 3xTg-ADmice •Cognitive deficits •Tau phosphorylation •Aβ •Synaptic proteins •Neuroinflammation •Insulin signaling	•Elevated Plus Maze •Open field •One-Trial Object Recognition Task •Accelerating Rotarod Test •Morris water maze	•Western blot analysis •Immunohistochemical staining •Levels of IR, insulin-like growth factor-1 receptor, insulin receptor substrate-1,PI3K, 3 phosphoinositide dependent protein Kinase-1, AKT, and GSK-3.	•The 3xTg-AD mice develop numerous NFTs after the age of 12 months, but hyperphosphorylation of tau occurs at earlier age. •Tau phosphorylate at multiple phosphorylation sites in the 3xTg-AD mice at theage of 7–8 months. •STZ treatment causes increase in tau hyperphosphorylation and neuroinflammation, a insulin signaling dysfunction, decrease in synaptic plasticity. •Intranasal insulin treatment will improve cerebral glucose metabolism and cognition.	[249]
13.	ICV-STZ 3 mg/kg unilaterally in right lateral ventricle Volume = 10 μl	AP: –0.8 mm ML: 1.5 mm DV: –3.6 mm	•Insulin signaling •Intranasal insulin •Tau hyperphosphorylation •Microglial activation	•Open field test •Morris water maze	•Western blotting •Immunohistochemical staining •Doublecortin evaluation •Evaluation of tau kinases, GSK-3, cdk5 and its activator p35, MAPK/ERK, JNK, and calcium/calmodulin- dependent protein kinase II	•Intranasal delivery of insulin is a non-invasive technique that bypasses the blood-brain barrier and delivers insulin from the nasal cavity to the CNS via intraneuronal pathway. •Intranasal insulin restore the dysregulation of tau kinases in the hippocampus of ICV-STZ rats	[250]
14.	ICV-STZ 3 mg/kg bilaterally Volume = 10 μl in both ventricles	AP: 0.8 mm ML: 1.5 mm DV: 3.6 mm	•AD •LX2343 •Cognitive deficits •Oxidative stress •Tauopathy •Mitochondria •GSK-3β inhibitor •Neuroprotection	•Morris water maze test	•Mitochondrial membrane potential assay- mitochondrial membrane potential was determined •Mitochondrial function assay- concentration of ATP was measured •Transmission electron microscopy-based assay •GSK-3β enzymatic activity assay •Western blot-Cytochrome c estimationTUNEL assay- Cell death in animal brain tissue was detected •Immunohistochemistry- detect theexpression of P396-Tau protein	•Protein levels of PSD95, synaptophysin, and vesicle-associated membrane protein 2, which are crucial for neurotransmission and synaptic plasticity, were detected by western blot. •Protective effect of LX2343 involves JNK/p38 pathway inhibition.	[251]
15.	ICV-STZ 3 mg/kg unilaterally in right lateral ventricle Volume = 5 μl	AP: 1.0 mm ML: 0.5 mm DV: 2.5 mm	•AD •Liraglutide •Tau neurofilaments •Streptozotocin	•Morris water maze test	•Western blot analysis-Protein concentration were measured •Microtubule binding assay •Immunohistochemistry staining-Detect NF and tau phosphorylation •Immunofluorescence staining- phosphorylation of NF-M/H detected by antibody SMI31	•Protein concentrations of the samples were measured by BCA Protein Assay Kit. •Microtubule binding assay tells about amount of tau and tubulin	[252]
16.	ICV-STZ 3 mg/kg bilaterally in both lateral ventricles	AP: –0.3 mm ML: –1.0 mm DV: –2.5 mm	•AD •Astrocyte activation •FPR2 •Tau phosphorylation	•Accelerating Rotarod test •Open field •Morris water maze	•Western blot- Protein concentrations were determined •Immunofluorescence staining	•FPR2 is known to be involved in host defense and inflammation. •As a receptor for Aβ, FPR2 could uptake and clear Aβ, suggesting a protective effect in brain. After being activated by Aβ, FPR could also induce glial cells to release proinflammatory factors, indicating its harmful effect on brain. •FPR2 deletion could improve cognitive function, hyperphosphorylation of tau, and the activation of astrocytes	[253]
17.	ICV-STZ 3 mg/kg bilaterally Volume = 2 μl/ventricle	AP: 0.8 mm ML: 1.5 mm DV: 3.6 mm	•Memory deficit •Tau hyperphosphorylation •GSK-3β •PP2A •Streptozotocin •Hippocampus	•Autoshaping Learning Task •Food Magazine and Autoshaping Training	•Western blot-levels of p-tau, levels of p-GSK-3β estimated	•Insulin and IR are selectively distributed in the brain, including olfactory bulb, hypothalamus, cerebral cortex, amygdala and hippocampus. •IR expression is involved in memory formation. •Binding of insulin to IR induces activating signal transduction cascade of the PI3K pathway in turn activates Akt/PKB, which phosphorylate the GSK-3 results in its inactivation causing tau hyperphosphorylation. •Disruption of IR-PI3K-Akt/PKB signaling cascade leads to the dephosphorylation in Ser9 of GSK-3β confirming GSK-3β, a major kinase that phosphorylate in vivo tau in several sites; hyperphosphorylated in PHF.	[254]
18.	ICV-STZ 3 mg/kg bilaterally Volume = 1 μl/ min	AP: 0.8 mm ML: 1.4 mm DV: 3.6 mm	•Nicotinamide •AD •SLN •Tau protein •PS	•Spatial learning and memory test	•ELISA tests- calculate the total tau protein amount (T-tau) and phosphorylated tau 231 •Histopathology of animals brains-cresyl violetstaining used	•Nicotinamide is ahistone deacetylase inhibitor. •The i.p. administration of nicotinamide loaded PS-SLN showed better memory improvement, preserved more neuronal cells and reduced the tau hyperphosphorylation in experimented animals comparing to its non-formulated conventional administration in the early stage of AD	[255]
19.	ICV-STZ 3 mg/kg bilaterally Volume = 5 μl/ventricle	AP: 0.8 mm ML: 1.5 mm DV: 3.6 mm	•TMP •Memory impairment •GSK-3β •Tau hyperphosphorylation •Cholinergic neuron	•Inhibitory avoidance task assay •Morris water maze assay	•Western blot-Total protein concentration was determined •Analysis of cholinergic function- ChAT and AChE activity was measured spectrophotometrically	•ICV-STZ, a good sporadic AD model causes spatial memory and fear memory impairments, impaired insulin signaling and overactivation of GSK-3β. •Activation of GSK-3β accounts for memory impairment, tau hyperphosphorylation, increased Aβ production, and neuroinflammation, all of which are hallmark characteristics of AD. •TMP was shown to decrease GSK-3β expression at both protein and RNA levels, and its analogue CXC195 could protect against cerebralischemia/reperfusion induced apoptosis through the PI3K/Akt/GSK-3β pathway.	[256]

4-HNE, 4-hydroxyl-2-nonenal; AChE, acetylcholinesterase; AD, Alzheimer’s disease; AKT, protein kinase B; AMPK, AMP-activated protein kinase; APP, amyloid-β proteinprecursor; Aβ, amyloid-β; cdk5, cyclin-dependent kinase 5; ChAT, choline acetyltransferase; CNS, central nervous system; ERK, extracellular signal regulated kinase; FPR2, formyl peptide receptor 2; GSK, glycogen synthase kinase; ICV-STZ, intracerebroventricular streptozotocin; IDE, insulin degrading enzyme; IR, insulin receptor; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated proteinkinase; MDA, malondialdehyde; NF, neurofilaments; NFTs, neurofibrillary tangles; PHF, paired helical filaments; PI3K, phosphatidylinositide 3-kinase; PP2A, protein phosphatase 2A; PPARγ, peroxisome proliferator-activated receptor gamma; PS, phosphatidylserine; PSD, postsynaptic density protein; ROS, reactive oxygen species; SLN, solid lipid nanoparticle; SOD, superoxide dismutase; TEM, transmission electron microscopy; TMP, tetramethylpyrazine.