Table 1.
Studies of pharmacological therapies with immunomodulatory properties in animal models of Alzheimer’s disease (AD)
| Study | Details |
|---|---|
| Fasudil | |
| Reference | Yu et al. (2017) |
| Number of animals, gender, ages, and treatment | Adult male amyloid precursor protein (APP)/presinilin 1 (PS1) transgenic (Tg) mice (APPswe/PSEN1deltaE9), 8 months of age, treated with Fasudil intraperitoneally (i.p.) (25 mg/kg per day, n = 8) for 2 months. Behavior was tested by Morris water maze (MWM) test. Animals were anesthetized and brains removed for biochemical analysis and immunohistochemistry (IHC). |
| Comparison | APP/PS1 Tg mice treated with saline i.p. (n = 8) for 2 months. Age-matched male wild type (WT) also served as control. |
| Functional outcomes | In MWM test, APP/PS1 Tg mice exhibited increased latency to target, latency to 1st entrance to southwest zone and mean distance to target compared to WT mice, suggesting that the learning and memory deficits in APP/PS1 Tg mice appeared. Significantly shorter time and distance spent by mice from the starting point on to the platform zone were observed in APP/PS1 + Fasudil mice compared with APP/PS1 + saline mice. APP/PS1 + Fasudil mice spent significantly greater time in southwest zone and distance in southwest zone. Using IHC to determine the amyloid-β (Aβ)42 expression in the hippocampus, the area of immunoreactive deposits of Aβ42 was decreased in APP/PS1 + Fasudil mice compared with APP/PS1 + saline mice. Also by Western blot assay, Fasudil significantly inhibited the levels of Aβ42 in the brains of APP/PS1 + Fasudil mice compared with APP/PS1 + saline mice. By IHC, the number of p-Tau/Ser396-positive cells in the hippocampus was significantly reduced in APP/PS1 + Fasudil mice compared with APP/PS1 + saline mice. Analysis by Western blot showed that expression of p-Tau/Ser396 protein was increased in APP/PS1 + saline mice compared with WT mice and there was a significant decrease in APP/PS1 + Fasudil mice compared with APP/PS1 + saline mice. IHC revealed a marked increase in the number of β-secretase (BACE)-positive cells in the hippocampus of APP/PS1 + saline mice compared with WT mice, and that Fasudil treatment partially attenuated this increase. Western blot assay indicated that expression of BACE protein in APP/PS1 Tg mice was significantly elevated compared with WT mice and was significantly decreased by Fasudil treatment for 8 weeks. Postsynaptic density-95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability and certain types of memory regulated by Aβ. By IHC, the number of PSD-95-positive cells in the hippocampus of APP/PS1 + saline mice was decreased compared with WT mice, while treatment of APP/PS1 Tg mice with Fasudil increased the expression. Using Western blot assay, the expression of PSD-95 was significantly higher in APP/PS1 + Fasudil mice compared with APP/PS1 + saline mice. By IHC and Western blot for cortex and hippocampus, p-nuclear factor-kappa B (NF-κB)/p65 expression was increased in APP/PS1 + saline mice compared with WT mice and Fasudil treatment decreased p-NF-κB/p65 expression, which was co-located in CD11b+ microglia. The levels of Rho-associated protein kinase-II (ROCK-II), Toll-like receptor (TLR)-4 and myeloid differentiation primary response 88 (MyD88) proteins were increased in APP/PS1 + saline mice compared with WT mice and Fasudil treatment significantly inhibited the expression of ROCK-II, TLR-4 and MyD88 proteins compared with APP/PS1 + saline mice. However, there was no significant difference in the expression of TLR-2 protein among the three groups. The levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in hippocampus were higher in APP/PS1 + saline mice compared with WT mice. Fasudil treatment significantly decreased the production of IL-1β, IL-6 and TNF-α, and significantly increased the production of IL-10 and interferon (IFN)-γ compared with APP/PS1 + saline mice. |
| Conclusion | Fasudil improved memory, reduced Aβ deposition and tau phosphorylation, decreased BACE, increased PSD-95 and inhibition of TLR-NF-κB-MyD88 inflammation and decreased production of proinflammatory cytokines IL-1β, IL-6 and TNF-α with increased production of anti-inflammatory cytokine IL-10 in APP/PS1 mice. |
| LW-AFC (a formula derived from Liuwei Dihuang Decoction) | |
| Reference | Wang et al. (2017) |
| Number of animals, gender, ages, and treatment | Adult male SAMP8 mice (senescence-accelerated mouse prone 8), 6 months of age, received intragastric administration of LW-AFC (0.1 mL/10 g body weight, 80, 160 and 320 mg/mL in water, n = 9–11) once a day for 150 days. After administration for 3 months, behavioral tests performed. Following the behavioral studies, blood plasma, hypothalamus, pituitary and spleen were collected for analysis. A graded scoring system is used for evaluation of degree of senescence in SAMP8 mice and was designed to assess changes in behavior and appearance of the mice. Grade 0 represented no particular changes and grade 4 represented the most severe changes (Hosokawa et al., 1984). |
| Comparison | SAMP8 mice as model group and SAMR1 mice (senescence-accelerated mouse-resistant 1) (n = 10) as control given an equal volume of water. |
| Functional outcomes | SAMP mice are a model of age-related/late-onset AD (Cheng et al., 2014), while SAMR1 mice represent a normal aging control (Shimada and Hasegawa-Ishii, 2011). The grading score for evaluation of degree of senescence in SAMP8 mice was significantly higher than in SAMR1 mice, and the grading score was significantly reduced after being treated with medium dosage LW-AFC (1.6 g/kg) while there was no significant difference between other treated groups and model group. The total distance of SAMP8 mice in the spontaneous locomotor test was significantly decreased compared with SAMR1 mice. SAMP8 mice administered LW-AFC (0.8, 1.6, 3.2 g/kg) had significantly increased locomotor activity compared to model group. The short (1 hour) and long term (24 hours) object recognition memory were deficit in SAMP8 mice compared to SAMR1 mice, while that of all the treated SAMP8 mice improved and administration with LW-AFC 1.6 g/kg significantly reversed the deficit. In MWM test, SAMP8 mice showed longer escape tendency than SAMR1 mice on the final day, and the latencies of SAMP8 mice treated with LW-AFC (0.8, 1.6 g/kg) were significantly less than the model group, thereby showing that LW-AFC (0.8, 1.6 g/kg) could ameliorate the impairment of spatial learning in SAMP8 mice. Biochemical analysis showed that in hypothalamic-pituitary-adrenal (HPA) axis the concentrations of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and corticosterone were significantly increased in SAMP8 mice compared to SAMR1 mice, while the level of CRH was significantly decreased by LW-AFC) (1.6, 3.2 g/kg), and ACTH was significantly decreased by LW-AFC (0.8, 1.6 g/kg). In hypothalamic-pituitary-gonadal (HPG) axis, the concentrations of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were significantly increased and testosterone (T) was significantly decreased in SAMP8 mice compared with SAMR1 mice, while the level of GnRH, FSH and LH was significantly decreased by LW-AFC (0.8, 1.6 g/kg) and T was significantly increased by LW-AFC (0.8, 1.6, 3.2 g/kg). These data show LW-AFC ameliorates imbalance of HPA and HPG axis in SAMP8 mice. Using multiplex bead analysis of blood plasma, IL-1β, IL-2, IL-6, IL-23, granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, chemotactic factor exotoxin, regulated on activation in normal T-cell expressed and secreted (RANTES), TNF-α and TNF-β were significantly increased, while IL-4 and IL-10, chemotactic factor monocyte chemotactic protein 1 (MCP-1) were significantly decreased in SAMP8 mice compared to SAMR1 mice. LW-AFC treatment decreased IL-1β, IL-2, IL-23, GM-CSF, IFN-γ, exotoxin, TNF-α and TNF-β and increased MCP-1 production in 0.8, 1.6, 3.2 g/kg dosage, and decreased IL-6 and RANTES in 0.8, 1.6 g/kg dosage, and meanwhile increased IL-5 production in 1.6 g/kg dosage. These results show LW-AFC administration regulated and restored the aberrant immune function in SAMP8 mice. |
| Conclusion | LW-AFC ameliorated cognitive deterioration and restored the imbalance in HPA and HPG axis, and regulated the abnormal production of cytokines in SAMP8 mice. |
| Reference | Wang et al. (2016) |
| Number of animals, gender, ages, and treatment | Adult male APP/PS1 mice, 9 months of age, received intragastric administration of LW-AFC (0.1 mL/10 g body weight, 160 mg/mL in water, n = 10–11) once a day for 150 days. Behavioral tests were performed starting at 102 days (locomotor activity test) from the beginning of LW-AFC administration and the last one at 131 days. Following the behavioral studies, blood plasma, hypothalamus, pituitary and spleen were collected for analysis. |
| Comparison | APP/PS1 mice as model group and age-matched male WT mice (n = 15) as control given an equal volume of water. |
| Functional outcomes | The locomotor activity test assessed the spontaneous motor activity of APP/PS1 mice, and no significant difference was found between the groups. The novel object recognition test assessed the object recognition memory of mice. LW-AFC treatment in APP/PS1 mice significantly decreased the preferential index, indicating that LW-AFC ameliorated the object recognition memory deficit of APP/PS1 mice. The MWM test assessed the spatial learning and memory of APP/PS1 mice. For the learning task, APP/PS1 mice had longer escape latencies than WT mice on the final test day and the latencies of LW-AFC treated APP/PS1 mice were significantly less than those of the model group. These results showed that LW-AFC ameliorated the spatial learning impairment of APP/PS1 mice. In the probe trial, the escape latency was increased, the number of plate crossings decreased and the time in the target quadrant was decreased, but swimming speed was not significantly different for APP/PS1 mice compared to WT mice. The escape latency decreased, the number of plate crossings increased, and the time in the target quadrant increased in APP/PS1 mice treated with LW-AFC. These findings indicated that LW-AFC significantly improved the spatial learning and memory deficits of APP/PS1 mice. LW-AFC also improved the passive avoidance impairment of APP/PS1 mice. Nissl staining showed typical neuropathological changes in the CA1 and CA3 regions of hippocampus in APP/PS1 mice compared to WT mice, including neuronal loss and nucleus shrinkage or disappearance. Significantly lower Nissl body numbers were seen in the whole brain, hippocampus, and CA1 and CA3 regions of APP/PS1 mice than in WT mice. LW-AFC treatment significantly decreased these neuropathological changes and increased the density of healthy neurons in the hippocampus and CA3 region of APP/PS1 mice. Thus, LW-AFC protected against neuronal loss in the hippocampus of APP/PS1 mice. APP/PS1 mice developed a significant number of Aβ plaques in the brain at 14 months, while Aβ plaques were not found in WT mice. LW-AFC treated APP/PS1 mice had a significantly smaller number of Aβ deposits in the whole brain and hippocampus. Hence, LW-AFC alleviated Aβ deposition in the brain of APP/PS1 mice. The levels of Aβ42 and Aβ40 in the hippocampus and blood plasma of APP/PS1 mice were significantly higher than for WT mice. LW-AFC treatment of APP/PS1 mice led to significantly lower Aβ42 levels in the hippocanpus and in the plasma than in WT mice. Within the HPA axis, the concentration of CRH, ACTH and corticosterone were significantly higher in APP/PS1 mice than in WT mice. LW-AFC significantly decreased the CRH and ACTH level. Within the HPG axis, the concentration of GnRH, FSH and LH were higher in APP/PS1 mice, but T was not significantly different between APP/PS1 and WT mice. LW-AFC significantly decreased the concentration of GnRH, FSH and LH in APP/PS1 mice. Significantly fewer CD8+CD28+ T cells and significantly more CD3+CD2+Foxp3+ T cells were observed in APP/PS1 mice than in WT mice. LW-AFC treatment of APP/PS1 mice increased the expression of CD8+CD28+ T cells and decreased that of CD3+CD2+Foxp3+ T cells. These findings showed that LW-AFC treatment partially restored nomal lymphocyte expression in APP/PS1 mice. By multiplex bead analysis of blood plasma, increases in the levels of IL-1β, IL-2, IL-6, IL-23, GM-CSF, TNF-α, TNF-β, eotaxin, and decreases in the levels of IL-4 and granulocyte-colony stimulating factor (G-CSF) were seen in APP/PS1 mice compared to WT mice. The levels of of IL-1β, IL-2, IL-6, IL-23, GM-CSF, TNF-α, TNF-β, eotaxin were decreased, and IL-4 and G-CSF were increased, after LW-AFC treatment. These results showed that cytokine secretion was abnormal in APP/PS1 mice and that administration of LW-AFC restored this aberrant immune function in APP/PS1 mice. |
| Conclusion | LW-AFC ameliorated behavioral and pathological deterioration, and restored the imbalance in HPA and HPG axis, and the aberrant immune function in APP/PS1 mice. |
| Curcumin | |
| Reference | Sundaram et al. (2017) |
| Number of animals, gender, ages, and treatment | p25 single Tg mice were crossed with Ca2+/calmodulin-dependent protein kinase IIα single Tg mice to generate bi-transgenic offspring (p25Tg mice) that inducibly overexpress the human p25 gene under the control of the Ca2+/calmodulin-dependent protein kinase promoter-regulated tet-off system. p25Tg mice were maintained on deoxycycline (200 µg/mL, in drinking water) from conception to 6 weeks postnatal to avoid any posible developmental consequences from the p25 expression. Hemizygous mice either male or female were used. p25 expresion was induced in 6-week-old mice by removal of deoxycycline in water and concurently treated with an optimized curcumin formulation, Longvida, orally via their feed (4 g/kg, 0.8 g/kg curcumin) of chow for 12 weeks. Behavioral studies were performed for 12-week induced (18-week-old) p25Tg and control mice with and without curcumin treatment (n = 5–6/group). Also biochenical assays were carried out on brain samples from p25Tg and control mice with and without curcumin treatment (n = 3/group). |
| Comparison | WT littermates were used as control. |
| Functional outcomes | Using IHC and Western blot with anti-green fluorescent protein (GFP) antibody, equivalent levels of p25 expression were confirmed in the curcumin-treated as well as non-treated p25Tg mice. There was no obvious change in cyclin-dependent kinase 5 (Cdk5) protein levels between the 12-week induced curcumin-treated and non-treated p25Tg mice. However, p-25-mediated Cdk5 hyperactivity was significantly decreased in curcumin-treated 12-week induced p25Tg mice compared to non-treated p25Tg mice. By IHC, the intensity of glial fibrillary acidic protein (GFAP) staining was reduced in the cortex and hippocampus of curcumin-treated 12-week induced p25Tg mice compared to non-treated p25Tg mice. Western blot analysis of GFAP levels showed there was a 2–3-fold significant decrease in GFAP expression in the forebrain of curcumin-treated p25Tg mice compared to non-treated p25Tg mice. Western blot analysis and cPLA2 activity assay results showed an approximately 3-fold significant decrease in p25-mediated cPLA2 upregulation in curcumin-treated 12-week induced p25Tg mice. Mass spectrometry data indicated that lysophosphatidylcholine (LPC) levels were significantly decreased by curcumin treatment in p25Tg mice. With IHC, altered immunostaining pattern with anti-CD11b (a microglial activation marker) was observed in both cortex and hippocampus of curcumin-treated 12-week induced p25Tg mice compared to non-treated p25Tg mice. Western blot analysis indicated a modest reduction in microglial activation in curcumin-treated 12-week induced p25Tg mice but was not significant with the small group sizes used. By real time polymerase chain reaction, the predominantly anti-inflammatory cytokine transforming growth factor-β levels were unaltered in curcumin-treated p25Tg mice. However, the proinflammatory cytokines macrophage inflammatory protein-1α, TNF-α, IL-1β expression levels in p25TG mice were significantly downregulated by curcumin treatment. Using IHC to study tau hyperphosphorylation levels, ATB (tau) immunostaining levels were decreased in curcumin-treated p25Tg mice compared to non-treated p25Tg mice. Western blot analysis showed a significant 2-fold reduction in ATB expression levels in curcumin-treated p25Tg mice. Histochemical staining assays showed a marked decrease in Aβ42 immunostaining of cortex and hippocampus in curcumin-treated p25Tg mice compared to non-treated p25Tg mice. Forebrain atrophy and neuronal apoptosis were apparent in 12-week induced p25Tg mice. IHC analysis showed cleaved caspase-3 immunostaining was reduced in cortex and hippocampus of 12-week induced p25Tg mice after curcumin treatment, indicating that curcumin confers neuroprotection against p25-induced neuronal death. Spatial memory was assessed using radial arm maze analyses and curcumin-treated p25Tg mice displayed better performance compared to non-treated p25Tg mice. Working memory errors were reduced almost to normal levels and reference memory errors decreased in curcumin-treated p25Tg mice. Hence, curcumin had a neuroprotetive capability to restore p25-inducd cognitive deficits in p25Tg mice. |
| Conclusion | Curcumin ameliorated neuroinflammation, neurodegeneration, and memory deficits in p25Tg mice. |
| Reference | Bassani et al. (2017) |
| Number of animals, gender, ages, and treatment | Adult male Wistar rats, 3-4 months of age, randomly divided into groups: streptozotocin (STZ, n = 7), STZ + curcumin 25 mg/kg (STZ + cu 25, n = 6), STZ + curcumin 50 mg/kg (STZ + cu 50, n = 8), STZ + curcumin 100 mg/kg (STZ + cu 100, n = 7). The animals in STZ groups received a single bilateral intracerebroventricular injection of STZ (3 mg/kg total dose) in saline (4.5 µL/injection site). Treatment with curcumin (25, 50 and 100 mg/kg, per os (p.o.)) or its vehicle (0.5% carboxymethylcellulose in water with 1% Tween 80) was performed over 30 days, once daily in afternoon, and started 1 hour before the beginning of stereotactic surgeries. Behavioral evaluations were started at 3 weeks after surgeries. The animals were assessed in the open field test to evaluate spontaneous locomotor activity and exploratory behavior on day 21 after surgery (day 0 = day of surgery) and in the elevated plus maze (EPM) on day 22 to assess anxiety-like behavior. Cognitive performance was evaluated in the object location test (OLT) on day 28, in the object recognition test on day 29, and in the spatial version of the Y maze on day 30. Immediately after the last behavioral analysis, blood glucose levels were measured using blood samples collected by tail prick. Afterwards animals were anaesthetized and intracardially perfused for IHC evaluation of brains. |
| Comparison | Sham treated animals served as control (n = 7). |
| Functional outcomes | The analysis of spontaneous locomotor activity in the open field test did not reveal any changes in the locomotor parameters analysed in all of the groups compared to the sham group. In the EPM, a decrease in the time spent in closed arms and percentage of time spent in closed arms was seen in the STZ and STZ + cu 100 groups compared to the sham group, which is an indication of decreased anxiety-like behavior in these animals. None of the groups showed more anxiety-like behavior than the sham group in all of the EPM parameters tested. Anxiety-like behavior can reduce the exploration of novelty in the OLT, object recognition test, and Y maze. There were no significant alterations in blood glucose level in all of the groups compared with the sham group. Elevated blood glucose is another factor that could interfere with cognitive performance. The STZ animals showed deficits in short-term spatial memory, reflected in a decrease in the discrimination index in the OLT and a decrease in the time spent on the new arm in the Y maze when compared with the sham group. In the OLT, none of the curcumin-treated groups showed an increased discrimination index compared with the STZ group. In the Y maze, none of the curcumin-treated groups spent more time in the new arm than the STZ group, nor was the time spent on the new arm greater than 33%. Therefore treatment of STZ-lesioned animals with curcumin in different doses did not prevent the impairment in short-term spatial memory. In the object recognition test, the STZ animals exhibited deficits in short-term recognition memory, reflected by a decrease in the discrimination index compared with the sham group. The STZ + cu 50 and STZ + cu 100 groups exhibited an increased discrimination index compared with the STZ group, thereby suggesting a possible beneficial effect of curcumin on short-tem recognition memory at these doses. The STZ animals exhibited decreased newborn neurons in both the subventricular zone of the lateral ventricles (LVs) and subgranular zone of the dentate gyrus (DG) of the hippocampus, reflected by decreased doublecortin (DCX) immunoreactivity (IR) in the subventricular zone of the LVs and decreased DCX-positive cells in the DG of the hippocampus compared with the sham group. Treatment with curcumin in any dose did not restore DCX-IR in the subventricular zone and DCX-positive cells in the DG when compared with the STZ group. At 30 days after surgery, the STZ group showed a marked increase in ionized calcium binding adaptor molecule-1 (Iba-1)-IR in all periventricular areas analysed (i.e. LVs, septum and corpus callosum) and also in the CA1 and CA2 areas of the dorsal hippocampus, but not in the DG, when compared with the sham group. Curcumin treatment at 25 and 50 mg/kg caused an improvement in Iba-1-IR in corpus callosum but not in the LVs and septum, despite an important but not significant reduction in the LVs and septum. In the hippocampus, no improvement in Iba-1-IR was found in the DG, CA1 and CA2 areas for all of the curcumin doses tested. Similarly, the STZ animals showed a marked increase in GFAP-IR in all periventricular areas analysed (i.e., LVs, septum and corpus callosum) and also in the DG, CA1 and CA2 areas of the dorsal hippocampus when compared with the sham group. Curcumin treatment did not improve GFAP-IR in the periventricular areas and the hippocampus with all of the doses tested. |
| Conclusion | Curcumin improved short-term recognition memory and exerted only slight improvement in neuroinflammation, resulting in no improvement in hippocampal and subventricular neurogenesis. |
| Soluble TNF (sTNF) inhibitor XPro1595 | |
| Reference | MacPherson et al. (2017) |
| Number of animals, gender, ages, and treatment | Female 5xFAD mice were used for all flow cytometry, XPro1595, and electrophysiology experiments. Female mice show accelerated AD-like pathology when compared to male 5xFAD mice. For measurement of cytokines in cerebrospinal fluid and inflammatory mRNA analysis, both male and female mice at 2, 4 and 6 months of age were used. To assess the role of soluble TNF in AD-like pathology and immune cell trafficking in the CNS, Tg female mice were treated subcutaneous (s.c.) with XPro1595 (10 mg/kg) twice weekly for 2 months. To assess the role of sTNF on trafficking immune cell population, female mice were treated between 5 and 7 months of age with XPro1595 (n = 10). A second cohort of mice was treated between 2 and 4 months of age with XPro1595 (n = 7). To assess the role of sTNF on AD-like amyloid accumulation and mRNA associated with inflammatory markers, female Tg and non-Tg mice were treated from 5 to 7 months of age with XPro1595 (Tg XPro1595 n = 5, non-Tg XPro1595 n = 6). Brains were removed for IHC and histological analysis. |
| Comparison | Female Tg mice at 2, 4 and 6 months of age were treated s.c. with saline vehicle twice weekly for 2 months as control. Female mice were treated between 5 and 7 months of age with saline as control (n = 9). Other female mice were treated between 2 and 4 months of age with saline (n = 8). Female Tg and non-Tg mice were treated from 5 to 7 months of age with saline (Tg saline n = 3, non-Tg saline n = 8). |
| Functional outcomes | The 5xFAD Tg mouse model of AD shows progressive Aβ plague accumulation before 3 months of age, progressive synaptic protein loss as well as later neuronal degeneration, and cognitive deficits by 5 months of age (Oakley et al., 2006). In this study, male and female Tg mice were not found to express significantly different levels of several cytokines in cerebrospinal fluid at 2, 4 or 6 months of age. Levels of murine KC/chemokine (C-X-C motif) ligand 1 protein were significantly lower in Tg mice at 6 months of age when compared to non-Tg mice. Cerebrospinal fluid levels of IFN-γ, IL-10, IL-1β, IL-6, and IL-12p70 protein levels were undetectable. TNF protein levels were not detectable at 2 months of age and no significant differences were found at 4 and 6 months of age. Within the cortex, there was a significant increase in expression of CD45 mRNA at 6 months in Tg mice compared to non-Tg mice, male and female. Within the midbrain, expression of CD45 and TNF mRNA was significantly increased at 6 months in Tg mice compared to non-Tg mice. Immune cells were isolated from the CNS of Tg and non-Tg mice at 3.5, 5, 7 and 12 months of age. These ages correspond to early stages of amyloid deposition (3.5 months), onset of cognitive impairment (5 months), and aggressive amyloid burden (7 and 12 months) (Oakley et al., 2006). Peripheral macrophages that have trafficked into the brain are CD11b+ and CD45high. The CD11b+CD45high population was not purely peripheral macrophages but also contained activated microglia and dendritic cells, which also express CD11b and CD45 at high level. Within the brain there was no effect of genotype or age on the number of CD11b+CD45high cells, but within this population there was a significant decrease in the frequency of CD11b+-CD45highMHCII (major histocompatibility complex II)+ cells in Tg mice compared to non-Tg mice at 7 months of age. In Tg mice there was a significant increase in the ratio of CD11b+Ly6Chigh:Ly6Chigh at 5 months in the brain compared to non-Tg mice. In the brains of Tg and non-Tg mice, there were no significant differences in the frequency or number of neutrophils (CD3–Ly6G+), B cells (CD3–Ly6G–CD11b–CD19+), or dendritic cells (CD3–Ly6G–CD11b+CD11c+). At 12 months of age, the frequency of CD3+ T cells of all CD45+ brain cells was significantly increased in Tg mice compared to non-Tg mice. The number of CD4+ T cells was not significantly different between Tg and non-Tg mice; however, at 12 months of age there was a significant increase in the number of CD8+ T cells in the brains of Tg mice compared to non-TG mice. Tg mice showed alterations of the CD8+ effector (CD62L–CD44–) T cell population compared to non-Tg mice. At 3.5 months of age, there was a significant decrease in the frequency of CD8+ effectors, while at 5 months of age there was a significant increase in the number of CD8+ effectors in Tg mice compared to non-Tg mice. No significant changes were found in the other subpopulations of CD8+ or CD4+ T cells. Tg mice were treated s.c. with Xpro1595 (10 mg/kg) twice weekly for 2 months starting at 5 months of age or starting at 2 months of age to assess the role of sTNF on AD-like pathology after significant plaque deposition or as plaque deposition began to build up, respectively. Inhibition of sTNF with Xpro1595 between 5 and 7 months of age decreased MHCII+ microglia/macrophages within both populations of activated microglia/peripheral cells (CD11b+CD45high, frequency but not number) and quiescent/homeostatic resident microglia (CD11b+CD45low, frequency and number) immune cells. While these effects on MHCII+ populations were not observed in Tg mice between 2 and 4 months of age, there was an overall decrease in the frequency of CD11b+CD45high populations and an increase in CD11b+CD45low populations. Among other immune call populations in the brain, no changes were detected in frequency or number of B cells or neutrophils when XPro1595 was administered from 5 to 7 months of age in Tg mice. Within the dendritic cell population, there was a significant increase in frequency of CD11c+ cell population in the brain of Tg mice compared to non-Tg mice but there was no significant increase in cell number. When XPro1595 was administered to mice from 2 to 4 months of age, there was no significant increase in number or frequency of CD11c+ cells within the CD11b+ population. Within this population, only when XPro1595 was administered from 2 to 4 months of age but not from 5 to 7 months of age there was a significant decrease in the frequency of a MHCII+ population. Inhibition of sTNF by XPro1595 significantly decreased the density (% of area) of Aβ in the subiculum but not the entire hippocampus of Tg mice. Within the subiculum there was a significant decrease in Aβ density in the dorsal and posterior subiculum but not the ventral subiculum. No significant changes were found in the hippocampus or subiculum in the density of amino cupric silver stain which denotes disintegration of multiple neuronal elements including cell bodies, axons, dendrites and synaptic teminals. When the contralateral hemisphere was used to measure transcriptional changes in gene expression, gene expression associated with an inflammatory response and its resolution was found to be reduced by inhibition of sTNF with XPro1595. Transforming growth factor-β and chemokine (C-C motif) ligand 2 mRNA expression was significantly reduced in Tg mice, but not non-Tg mice, treated with Xpro1595 from between 5 and 7 months of age. Deficits in hippocampal CA1 synaptic function have been reported in several mouse models of AD and are associated with cognitive deficits. To determine if in vivo peripheral administration of XPro1595 modulates synaptic function in Tg mice, brain slices were harvested for analysis of CA3-CA1 synaptic strength curves and long-term potentiation (LTP) levels. At 4 months of age, basal synaptic strength deficits were found to be relatively mild in vehicle-treated Tg mice, characterized by a modest downward shift in the synaptic strength curve and a significant reduction in the maximal excitatory postsynaptic potential/fiber volley ratio, relative to vehicle-treated non-Tg mice. XPro1595 had minimal effect on synaptic strength, though it did slightly reduce and slightly increase the maximal excitatory postsynaptic potential/fiber volley ratio in non-Tg and Tg mice, respectively. Relative to vehicle-treated non-Tg mice, Tg mice showed a significant reduction in LTP levels at 60 minutes post-100 Hz stimulation that was blocked by in vivo treatment with XPro1595. In contrast, XPro1595 had minimal effect on LTP in non-Tg mice. |
| Conclusion | Administration of XPro1595 reduced the age-dependent increase in activated immune cells in Tg mice, while decreasing the overall number of CD4+ T cells. In addition, XPro1595 treatment in vivo rescued LTP measured in brain slices in association with decreased Aβ plaques in the subiculum. |
| IL-1 receptor antagonist (IL-1RA) | |
| Reference | Zou et al. (2016) |
| Number of animals, gender, ages, and treatment | APPswe/PSEN1deltaE9 mice were crossed with GFP-M mice to obtain double Tg offspring which were heterozygous for the corresponding genes (deltaE9+/– × GFP+/–). Adult female Tg mice (4–5 months of age were used. Mice were anesthetized, dexamethasone (6 mg/kg) was injected to prevent development of cerebral edema, and a piece of the skull was removed above the somatosensory cortex. The exposed brain was cleaned with sterile saline and covered with a round glass coverslip (D = 4 mm). The margin between the glass and skull was sealed with dental cement.Post-surgical mice were s.c. injected with carprofen (4 mg/kg) and cefotaxime (250 mg/kg). Lentiviruses encoding IL-1RA were intraparenchymally injected into the cortex before implanting the coverslip. Injection of lentiviruses (200 nL per time at a titre of ~1 × 108 infecting units/mL) was performed at 4 different sites in the exposed area of brain at a depth of 700–800 µm. After 4 weeks recovery period, apical dendrites originating from GFP-labeled layer V pyramidal neurons were imaged in consecutive sessions (once/week). GFP was excited by a femtosecond laser at 880 nm wavelength. To ensure the dendrites were chosen in amyloid plaque-free regions, methoxy-X04 (1 mg/kg) was injected i.p. 24 hours before imaging in the first and last time points (n = 4–6/group). |
| Comparison | GFP positive littermates without APP/PS1 transgenes were used as controls (deltaE9+/– × GFP+/–). |
| Functional outcomes | Replicating the preclinical stages of AD, 4–5 months old APPswe/PSEN1deltaE9 mice develop Aβdeposits without cognitive decline. In a previous study normal spine density and dynamics were observed far away from Aβ deposits in deltaE9 at this age (Zou et al., 2015). To assess if activity-induced structural spine plasticity on these dendrites was disturbed in preclinical AD, APP/PS1deltaE9 mice at 4–5 months of age were housed under enriched environment (EE) over 5 weeks and the apical tufts of layer V pyramidal neurons in the somatosensory cortex monitored. EE, which provides a spectrum of synaptic inputs and which leads to adaptive synaptic alterations within the adult brain, induced a steady increase of spine density in the control group. In contrast, EE failed to increase spine density in deltaE9 mice. Unlike control mice in which there was a gradual decline in dendritic spine elimination upon EE, the rate of spine elimination in deltaE9 mice remained unchanged. EE did not alter the rate of spine formation in both groups. IL-1β is a key mediator of the inflammatory response in AD and is known to have deleterious effects on synaptic plasticity (Tong et al., 2012). The expression of IL-1β was significantly increased in cortical tissue of deltaE9 mice. To diminish IL-1β activity, lentiviruses expressing IL-1RA was injected into the somatosensory cortex. IL-1RA rectified the adaptive gain of spines upon EE in deltaE9 mice, accompanied with the gradual decline in spine elimination instead of rising spine formation. |
| Conclusion | Anti-inflammatory treatment with IL-1RA in deltaE9 mice successfully rescued the impairment in increasing spine density during EE. |
| Pioglitazone | |
| Reference | Zou et al. (2016) |
| Number of animals, gender, ages, and treatment | APPswe/PSEN1deltaE9 mice were crossed with GFP-M mice to obtain double Tg offspring which were heterozygous for the corresponding genes (deltaE9+/– × GFP+/–). Adult female Tg mice 4–5 months of age were used. Pioglitazone (350 ppm) was supplemented into rodent chow. Cranial window implantation and in vivo imaging as described above in the study with IL-1RA (n = 4–6/group). |
| Comparison | GFP positive littermates without APP/PS1 transgenes were used as controls (deltaE9+/– × GFP+/–). |
| Functional outcomes | It is known that amyloid plaques are surrounded by activated glial cells that release proinflammatory cytokines (Watkins et al., 2001). To investigate if these cytokines caused the impaired adaptive plasticity, deltaE9 mice were treated with pioglitazone, a peroxisome proliferators-activated receptor-γ agonist which inhibits the production of proinflammatory cytokines without affecting synaptic plasticity (Jiang et al., 1998; Chen et al., 2015). Pioglitazone treatment p.o. over 6 weeks period rehabilitated the steady increase of spine density in deltaE9 mice during exposure to EE. As in control mice, the EE-induced spine density increase resulted from the gradual decline in spine elimination, while the rate of spine formation was unchanged. |
| Conclusion | Anti-inflammatory treatment with pioglitazone in deltaE9 mice successfully rescued the impairment in increasing spine density during EE. |
| Reference | Yu et al. (2016) |
| Number of animals, gender, ages, and treatment | Homozygous 3xTg AD mice with PS1M146V, APPSwe and tauP301L transgenes were used. Female 3xTg mice at 10 months of age were subjected to behavioral tests and then treated with an experimental diet containing pioglitazone hydrochloride for 4 months. The experimental diet contained 200 mg pioglitazone/kg of rodent chow, and unmodified chow was used as the control diet. The body weight of mice and food consumption were measured every week. After the drug treatment (at age of 14 months) the mice were subjected to behavioral tests again. Then the mice were euthanized and the brains removed and hippocampi and cerebral cortices dissected and frozen for biochemical analyses. Behavioral tests (n = 11–12/group) and biochemical and IHC analyses (n = 4–8/group) were performed. |
| Comparison | WT mice served as control. |
| Functional outcomes | The 3xTg AD mice had slightly smaller body weight than WT control mice. Treatment with pioglitazone started to show a slight reduction in body weight after treatment for 13 weeks, and this decrease became statistically significant after treatment for 16 weeks. This decrease in body weight was not seen in WT mice after treatment with the same dose of the drug for the same period of time. During the same period of time, the food consumption among the groups or treatments was not different. The 3xTg AD mice explored for a longer distance in the open field and showed a higher fall latency in the rotarod test than WT mice. These findings indicated that the 3xTg AD mice have a more active spontaneous exploratory activity and perform better in motor coordination and balance. Treatment of the 3xTg AD mice and WT control mice with pioglitazone for 4 months did not significantly alter the spontaneous exploratory activity or locomotion. At 10 months of age, the 3xTg AD mice spent significantly less time in the open arm of the elevated plus maze than did the WT mice, but they spent approximately double the time and traveled double the distance than the WT mice in the center of the open field. These observations suggest a decreased anxiety level of the 3xTg AD mice at 10 months of age. Treatment of 3xTg AD mice and WT mice with pioglitazone did not significantly alter the time spent in the open arm of the EPM or the time spent and distance traveled in the center of the open field. These results suggest that pioglitazone treatment did not affect the anxiety level of the mice. At the age of 10 months before the treatment, the 3xTg AD mice took a longer time and swam a longer distance than WT mice to find the escape platform during the training phase, although improvement from day to day was seen in the 3xTg AD mice. This suggested a learning impairment and it appeared more severe when the mice reached 14 months of age. Treatment of 3xTg AD mice with pioglitazone for 4 months improved the learning of the mice as evidenced by significantly shorter escape latency and swimming distance on day 4 to reach the escape platform than the untreated 3xTg AD mice. These results suggested that pioglitazone improved learning of the 3xTg AD mice. Probe trials were performed 1 day after the last training in the MWM to assess the spatial reference memory of the mice. An impairment of the spatial reference memory in the 3xTg AD mice was observed at both 10 and 14 months of age, as evidenced by less time spent and shorter distance swam within the target quadrant by the 3xTg AD mice than WT mice during the probe test. Pioglitazone treatment did not significantly affect the performance in the probe test, suggesting that treatment with pioglitazone for 4 months did not improve spatial reference memory. The total tau level in the hippocampus was several fold higher, but in the cerebral cortex only slightly higher, in the 3xTg AD mice than in WT mice. Treatment of the mice with pioglitazone for 4 months did not significantly alter the total tau level. Treatment with pioglitazone reduced tau phosphorylation at several AD-related sites in the hippocampus of both 3xTg AD and WT mice. This reduction was only seen at a few phosphorylation sites in the cerebral cortex. Treatment of both 3xTg AD and WT mice with pioglitazone for 4 months increased phosphorylation of AKT at Ser473 and of GSK3β at Ser9 in the hippocampus. These findings suggested that pioglitazone treatment promoted insulin-AKT signaling that in turn led to inhibition of GSK3β. No significant promotion of AKT and inhibition of GSK3β were found in the cerebral cortex. A significant increase of GFAP staining (a marker of astrocytes and neuroinflammation) was observed in the brains of the 3xTg AD mice compared with WT mice. Treatment with pioglitazone for 4 months reduced GFAP staining in the brains of both 3xTg AD and WT mice, suggesting that pioglitazone treatment may attenuate neuroinflammation. |
| Conclusion | Pioglitazone treatment improved spatial learning, enhanced AKT signaling, and decreased tau hyperphosphorylation and neuroinflammation in 3xTg AD mice. |
| Rosiglitazone | |
| Reference | Yu et al. (2015) |
| Number of animals, gender, ages, and treatment | Homozygous 3xTg AD mice with PS1M146V, APPSwe and tauP301L transgenes were used. Female 3xTg mice at 10 months of age were subjected to behavioral tests and then treated with an experimental diet containing rosiglitazone maleate for 4 months. The experimental diet contained 50 mg rosiglitazone/kg of rodent chow. After the drug treatment (at age of 14 months) the mice were subjected to behavioral tests again. Then the mice were euthanized and the brains removed and hippocampi and cerebral cortices dissected and frozen for biochemical analyses. Behavioral tests (n = 11–12/group) and biochemical and IHC analyses (n = 4–8/group) were performed. |
| Comparison | WT mice served as control. |
| Functional outcomes | Treatment with rosiglitazone started to show a slight reduction in body weight after treatment for 13 weeks, and this decrease became statistically significant after treatment for 16 weeks. This decrease in body weight was not seen in WT mice after treatment with the same dose of the drug for the same period of time. During the same period of time, the food consumption among the groups or treatments was not different. Treatment of the 3xTg AD mice and WT control mice with rosiglitazone for 4 months did not significantly alter the spontaneous exploratory activity or locomotion. Treatment of 3xTg AD mice and WT mice with rosiglitazone did not significantly alter the time spent in the open arm of the elevated plus maze or the time spent and distance traveled in the center of the open field. These results suggest that rosiglitazone treatment did not affect the anxiety level of the mice. Treatment of 3xTg AD mice with rosiglitazone for 4 months did not improve the learning of the mice or the spatial reference memory. Treatment of the mice with rosiglitazone for 4 months did not significantly alter the total tau level. Treatment of both 3xTg AD and WT mice with rosiglitazone for 4 months increased phosphorylation of AKT at Ser473 and of GSK3β at Ser9 in the hippocampus. These findings suggested that rosiglitazone treatment promoted insulin-AKT signaling that in turn led to inhibition of GSK3β. No significant promotion of AKT and inhibition of GSK3β were found in the cerebral cortex. A significant increase of GFAP staining (a marker of astrocytes and neuroinflammation) was observed in the brains of the 3xTg AD mice compared with WT mice. Treatment with rosiglitazone for 4 months reduced GFAP staining in the brains of both 3xTg AD and WT mice, suggesting that rosiglitazone treatment may attenuate neuroinflammation. |
| Conclusion | Rosiglitazone treatment enhanced AKT signaling, and decreased tau hyperphosphorylation and neuroinflammation in 3xTg AD mice. |
| Quercetin | |
| Reference | Sabogal-Guaqueta et al. (2015) |
| Number of animals, gender, ages, and treatment | Homozygous 3xTg AD and non-Tg mice, 18–21 months of age, were treated i.p. with quercetin 25 mg/kg in 0.1% dimethyl sulfoxide every 48 hours for 3 months. At 48 hours after the final treatment, behavioral tests (8–16 mice for each test) were performed. At 24 hours after the final behavioral test, the animals were anesthetized, perfused with saline and 4% paraformaldehyde, and the brains removed for IHC and histology (3–5 mice for each study) and biochemical analyses (4–6 mice for each analysis). |
| Comparison | 3xTg AD and non-Tg mice received i.p. injections of 0.1% dimethyl sulfoxide vehicle. |
| Functional outcomes | The subiculum showed a decrease of cell density in the vehicle treated 3xTg AD mice, and quercetin treatment increased the cell density in the subiculum to a level similar to that in non-Tg mice treated with vehicle or quercetin. No changes in cell density were observed in the other structures evaluated. These findings were supported by a significant loss of NeuN immunoreactivity in the subiculum of 3xTg AD mice treated with vehicle, which was prevented by quercetin treatment. NeuN immunoreactivity in the subiculum of the quercetin-treated 3xTg AD mice was similar to that in the non-Tg mice. The CA1 area of hippocampus, the entorhinal area and the amygdala did not show any alteration in NeuN immunoreactivity. The 3xTg AD mice showed strong Aβ immunoreactivity when compared with non-Tg mice 21–24 months of age. Quercetin treatment resulted in a significant decrease in the amount of extracellular Aβ deposition in all of the cerebral regions of the brain examined compared to vehicle treatment. The vehicle- or quercetin-treated non-Tg mice did not show Aβ immunoreactivity. A significant decrease was found in the levels of C-terminal APP fragments (β) in the quercetin-treated 3xTg AD mice, which were significantly lower than those in the vehicle-treated 3xTg AD mice. Also there was a significant reduction in Aβ40 and Aβ42 levels in the hippocamus of the quercetin-treated 3xTg AD mice compared to the vehicle-treated 3xTg AD mice. The vehicle-treated 3xTg AD mice had abundant ATB immunoreactivity whereas the quercetin-treated 3xTg AD mice displayed a significant decrease in these neurofibrillary tangles in the CA1 area, the subiculum and the amygdala but not in the entorhinal area. These findings were confirmed by a significant reduction in paired helical filament-1 and ATB protein levels in hippocampal and amygdalar lysates from quercetin-treated 3xTg AD mice compared to vehicle-treated 3xTg AD mice. The total tau level was not altered by any treatment. The 3xTg AD mice had a significant increase in GFAP immunoreactivity compared to non-Tg mice. However, quercetin treatment significantly reduced GFAP immunoreactivity in the CA1 area, the entorhinal area and the amygdala compared to vehicle treatment, but no changes were seen in the subiculum. Quercetin treatment did not affect the non-Tg mice. The quercetin-treated 3xTg AD mice had significantly reduced microglial activation in the CA1 area, the subiculum and the amygdala compared to vehicle-treated 3xTg AD mice, and was similar to that in the non-Tg mice. There were no changes in the entorhinal area. Quercetin treatment for 3 months of 18–21-month-old 3xTg AD mice significantly reduced the latency to locate the platform in the MWM test and improvement occurred in the spatial learning tasks after 8–10 trials. AD mice exhibit synaptic dysfunction and LTP deficits. In the EPM test, the non-Tg mice treated with quercetin or vehicle rarely visited the open arm, very similar to the 3xTg AD mice treated with vehicle, whereas the 3xTg AD mice treated with quercetin spent more time in the open arm. The time spent rearing was significantly lower in the quercetin-treated 3xTg AD mice than in the other mice. |
| Conclusion | Quercetin treatment decreased extracellular β-amylodosis, taupathy, astrogliosis and microgliosis in hippocampus and amygdala of 3xTg AD mice. In addition, quercetin improved performance on learning and spatial memory tasks and greater risk assessment behavior of the 3xTg AD mice in the EPM test. |
| IL-1β | |
| Reference | Rivera-Escalera et al. (2014) |
| Number of animals, gender, ages, and treatment | APP/PS1 mice, 7 months of age, were used. APP/PS1 mice were crossed to C-C chemokine receptor type 2 (CCR2)–/– mice, and then backcrossed to CCR2–/– mice to generate APP/PS1/CCR2–/– mice. APP/PS1 mice crossed to IL-1βXAT mice to produce APP/PS1-IL-1βXAT mice were used as recipients in a bonw marrow chimeric experiment. Mice were anesthetized, secured in a stereotactic apparatus and a 0.5 mm burr hole drilled in the skull and a 33 gauge needle pre-loaded with feline immunodeficiency virus (FIV)-Cre was lowered 1.8 mm from the brain surface over 2 minutes. A microsyringe pump controller was used to inject 1.5 µL of virus (~1.5 × 104 IVP) at a constant rate over 10 minutes. Following a 5 minutes delay to allow viral diffusion, the needle was raised slowly over 2 minutes, the burr hole sealed with bone wax and soft tissues sutured. APP/PS1 and APP/PS1-IL-1βXAT bone marrow chimeric animals received unilateral FIV-Cre injections at 7 months of age and were euthanized at 8 months of age for biochemical analysis of brain tissue. For experiments using APP/PS1 and APP/PS1/CCR2–/– mice, two 0.5 mm burr holes were drilled, one on each side, and a 33 gauge needle was lowered 1.5 mm over 2 minutes and 5 µL injected of recombinant adeno-associated virus vector 2 expressing IL-1β (rAAV2-IL-1β) or recombinant adeno-associated virus vector 2 (rAAV2) resulting in delivery of approximately 1.5 × 108 infectious particles/mL into each hippocampus. Following AAV2 delivery, 2 minutes was allowed for diffusion of viral particles. The needle was raised over 2 minutes and the burr hole sealed with bone wax. The procedure was then repeated to deliver the same viral vector on the opposite side. APP/PS1/CCR2–/– bone marrow chimeric animals received rAAV2-IL-1β into one hippocampus and rAAv2-Phe (a control viral vector) into the contralateral hippocampus at 9 months of age. All animals were euthanized 4 weeks post-viral transduction for brain tissue analysis. Group sizes were 4–12/group. For construction of bone marrow chimeras 8 to 12 week-old APP/PS1, APP/PS1-IL-1βXAT, or APP/PS1/CCR2–/– mice received two doses of 6 Gy total body irradiation separated by 4 hours. The head was shielded during the irradiation procedure to avoid confounding effects of cell recruitment and radiation-induced brain inflammation. Immediately after the second total body irradiation dose, APP/PS1, APP/PS1-IL-1βXAT, or APP/PS1/CCR2–/– mice were reconstituted with bone marrow derived from tibias and femurs of either GFP or GFP-CCR2–/– donor mice. Each bone marrow recipient received 200 µL of suspension for a total of 3 × 106 cells via tail vein injection. After a 6-week reconstitution period, APP/PS1 and APP/PS1-IL-1βXAT mice were subjected to tail bleeds for analysis of GFP positive leukocytes by flow cytometry. For APP/PS1/CCR2–/– mice, peripheral blood was collected at the time of euthanasia to determine bone marrow constitution and levels of monocyte subsets in the circulation. |
| Comparison | – |
| Functional outcomes | APP/PS1 or APP/PS1-IL-1βXAT mice received either WT-GFP or CCR2–/–-GFP bone marrow at 2–3 months of age. At 7 months of age all groups received unilateral hippocampal FIV-Cre injections and were euthanized at 8 months of age for analysis of GFP positive cells around amyloid plaques as well as Congo Red and 6E10 plaque indices. No GFP positive cells surrounded plaques without IL-1β overexpression. Significantly fewer GFP positive cells surrounded plaques in APP/PS1-IL-1βXAT mice that received CCR2–/–-GFP bone marrow. There was an overall effect of IL-1β on 6E10 labeled plaques and significant reductions in 6E10 plaques for both WT bone marrow recipients and CCR2–/– recipients following 1 month of IL-1β overexpression. There was a significant reduction in Congo Red plaques for both WT bone marrow recipients and CCR2–/– recipients. These findings suggested that peripheral mononuclear cells are recruited following IL-1β overexpression, but are not necessary for IL-1β-mediated amyloid plaque clearance. The 7-month-old APP/PS1 mice transduced intrahippocampally with rAAV2-IL-1β exhibited robust microglial activation 4 weeks following transduction compared to APP/PS1 mice transduced with rAAV2-Phe, a control vital vector. In addition to microglial activation, APP/PS1 mice transduced with rAAV2-IL-1β had increased levels of murine IL-1β and chemokine (C-C motif) ligand 2 detected in hippocampal tissue compared to APP/PS1 mice transduced with rAAV2-Phe. Transduction with rAAV2-IL-1β significantly reduced 6E10 and Congo Red staining of amyloid plaques in the hippocampus of APP/PS1 mice. Measurements of hippocampal Aβ peptide levels showed APP/PS1 mice transduced with rAAV2-IL-1β had significant decreases in levels of insoluble and soluble Aβ42 compared to APP/PS1 mice transduced with rAAV2-Phe. The neuroinflammation mediated by rAAV2-IL-1β in APP/PS1 mice dd not alter the levels of APP or its processing as the activity of BACE and its β-carboxy terminal fragment cleavage products remained unchanged. CCR2 signaling is important for the recruitment of CCR2+ monocytes to the brain (Saederup et al., 2010). APP/PS1 mice lacking CCR2 were generated as a way of ablating CCR2+ monocyte recrutment to the inflammed hippocampus. APP/PS1/CCR2–/– mice transduced with rAAV2-IL-1β had an increased inflammatory response 4 weeks following transduction as indicated by microglial activation and increased production of murine IL-1β and chemokine (C-C motif) ligand 2 when compared to APP/PS1/CCR2–/– mice transduced with control viral vector. Even in the absence of CCR2, rAAV2-IL-1β decreased 6E10 and Congo Red staining of amyloid plaques. Mesurements of hippocampal Aβ peptide levels showed APP/PS1/CCR2–/– mice transduced with rAAV2-IL-1β had significant decreases in insoluble Aβ42 and insoluble Aβ40 compared to control APP/PS1/CCR2–/– mice transduced with rAAV2-Phe. However, the levels of soluble Aβ42 and soluble Aβ40 were not significantly different from APP/PS1/CCR2–/– mice transduced with rAAV2-Phe. These findings confirmed that CCR2+ mononuclear cells are not necessary for IL-1β-mediated amyloid plaque clearanmce. |
| Conclusion | IL-1β overexpression in APP/PS1 mice ameliorated amyloid pathology, increased plaque-associated microglia, and induced recruitment of peripheral immune cells to the brain parenchyma. The IL-1β-mediated amyloid plaque clearance was independent of CCR2 signaling in the APP/PS1 mouse model of AD. |
| Cannabidiol | |
| Reference | Cheng et al. (2014) |
| Number of animals, gender, ages, and treatment | Adult male APPswe/PSEN1deltaE9 mice, 3 months of age, and their non-Tg WT littermates were treated daily late in the afternoon with a gel pellet containing cannabidiol (CBD) which they consumed within 2–5 minutes. CBD was used at a dose of 20 mg/kg body weight. CBD treatment was carried out for 5 months. Behavioral tests were performed starting at 10 months of age (n = 8–14/group). Mice were anesthetized, blood collected and centrifuged for plasma. Euthanized mice were perfused with phosphate buffer saline, and brains removed for biochemical analyses (n = 8–10). |
| Comparison | APPswe/PSEN1deltaE9 mice and WT littermates were given a vehicle gel pellet daily. |
| Functional outcomes | CBD treatment increased the time that APP/PS1 mice spent with the novel mouse in the social preference test, while no such effect was observed in WT mice, indicating that CBD had a beneficial effect on social recognition memory. In the EPM test, CBD treatment for 5 months had no effect on anxiety behaviors. In the associative learning test, APP/PS1 mice showed increased amounts of freezing at baseline regardless of treatment. CBD treatment had no effect on soluble and insoluble Aβ40 or Aβ42 in the cortex of APP/PS1 mice. Similarly, Aβ levels in the hippocampus were unchanged after CBD treatment. Total F2-isoprostanes (free and esterified corrected for arachidonic acid) were not significantly altered in APP/PS1 mice when compared to WT littermates. For enzymatically oxidised sterols, APP/PS1 mice exhibited significantly decreased overall levels of 24-hydroxycholesterol compared to WT littermates. No differences were found across all four groups for 27-hydroxycholesterol and the reactive species oxidised sterols, 7β-hydroxycholesterol and 7-ketocholesterol. Cholesterol was increased in cortical tissues of APP/PS1 mice compared to WT mice and CBD treatment increased cholesterol levels. There were no significant differences in the levels of mRNA for two inflammayory cytokine markers, IL-1β and TNF-α. No significant effect of CBD treatment on these cytokines was observed. All mice treated with CBD had significantly increased levels of CBD in blood plasma. |
| Conclusion | APP/PS1 mice developed a social recognition deficit that was ameliorated by CBD treatment. CBD treatment had no effect on anxiety or associative learning. The beneficial effect of CBD on social recognition memory was not associated with any changes in amyloid load or oxidative damage. |
| Memantine | |
| Reference | Borre et al. (2014) |
| Number of animals, gender, ages, and treatment | Adult male Sprague-Dawley rats, 240–270 g, 8–10 weeks of age, were anesthetized and two burr holes drilled and the olfactory bulbs aspirated through a blunt hypodermic needle (OBX animals). Group 3: OBX + vehicle (OBX-Veh) (n = 10); Group 4: OBX + memantine (OBX-Mem) (n = 10). Memantine (20 mg/2 mL/kg) or water (vehicle) was administered p.o. via gavage daily starting 2 days prior to OBX surgery and continued to 28 days after surgery. Behavioral tests were performed 7 to 18 days after OBX, with olfactory test at 29 days. Animals were euthanized at 30 days, and brains and spleens removed for analysis. |
| Comparison | Animals underwent sham surgery but without removal of olfactory bulbs. Group 1: sham operated + vehicle (Sham-Veh) (n = 10); Group 2: sham operated + memantine (Sham-Mem) (n = 10) |
| Functional outcomes | The observed weight loss in OBX animals was not due to decreased food consumption. OBX resulted in anosmia (complete loss of smell). The OBX-induced spatial memory deficit was reversed in T-maze by memantine treatment. Also the OBX-induced fear memory loss in the passive avoidance retention task was partly rescued by memantine treatment. Treatment with memantine attenuated the OBX-induced hyperactivity. The hippocampi of OBX animals fed the control diet weighed significantly less compared to sham controls, and memantine treatment attenuated the OBX-induced hippocampal atrophy. OBX resulted in a significantly lower cell count in the CA3, CA1 and DG areas of the dorsal hippocampus. Memantine treatment attenuated OBX-induced cell loss in the DG area but failed to rescue cell loss in the CA3 and CA1 areas. In the ventral hippocampus, OBX resulted in a significantly lower cell count in the CA3, CA1 and DG areas. Treatment with memantine rescued cell loss in the CA3, CA1 and DG areas. The presence of increased numbers of T cells in lymphoid organs signifies the induction of an immune response. OBX induces systemic inflammation (Song et al., 2009) and OBX increased the numbers of T cells in the spleen, suggesting the presence of systemic immune activation and possibly active inflammation. Treatment with memantine significantly decreased splenic T cells in OBX animals. |
| Conclusion | Memamtine treatment of OBX rats prevented/impeded the development of a neurodegenerative and depressive disorder and the concomitant cognitive deficits. |
| Multi-targeted diet | |
| Reference | Borre et al. (2014) |
| Number of animals, gender, ages, and treatment | Adult male Sprague-Dawley rats, 240–270 g, 8–10 weeks of age, were anesthetized and two burr holes drilled and the olfactory bulbs aspirated through a blunt hypodermic needle (OBX animals). Group 3: OBX + control diet (OBX-C) (n = 12); Group 4: OBX + Exp diet (OBX-Exp) (n = 12). Both diets were fed to the animals for 6 weeks (2 weeks prior to surgery and 4 weeks thereafter). The experimental diet was a control diet with supplements in mg/kg of food of zinc 1.63, curcumin 0.25, piperine 0.06, melatonin 0.03, choline 9.5, uridine 15.48, 3% soya + 4% tuna oil (25% docosahexaenoic acid/6% eicosapentaenoic acid). The relative calorific content of the two diets was approximately 4000 kcal/kg. |
| Behavioral tests were performed 7 to 22 days after OBX, with olfactory test at 29 days. Animals were euthanized at 30 days, and brains and spleens removed for analysis. | |
| Comparison | Animals underwent sham surgery but without removal of olfactory bulbs. Group 1: sham operated + control diet (Sham-C) (n = 12); Group 2: sham operated + experimental diet (Sham-Exp) (n = 12) |
| Functional outcomes | The observed weight loss in OBX animals was not due to decreased food consumption. OBX resulted in anosmia (complete loss of smell). The OBX-induced spatial memory deficit was reversed in T-maze, but not in the holeboard test, by the experimental diet. Also the OBX-induced fear memory loss in the passive avoidance retention task was partly rescued by the experimental diet. The experimental diet attenuated the OBX-induced hyperactivity. The hippocampi of OBX animals fed the control diet weighed significantly less compared to sham controls, and the experimental diet attenuated the OBX-induced hippocampal atrophy. OBX resulted in a significantly lower cell count in the CA3, CA1 and DG areas of the dorsal hippocampus. The experimental diet attenuated OBX-induced cell loss in the DG area but failed to rescue cell loss in the CA3 and CA1 areas. In the ventral hippocampus, OBX resulted in a significantly lower cell count in the CA3, CA1 and DG areas. The experimental diet rescued cell loss in the CA3, CA1 and DG areas. The presence of increased numbers of T cells in lymphoid organs signifies the induction of an immune response. OBX induces systemic inflammation (Song et al., 2009) and OBX increased the numbers of T cells in the spleen, suggesting the presence of systemic immune activation and possibly active inflammation. The experimental diet significantly decreased splenic T cells in OBX animals. |
| Conclusion | A diet targeting multiple disease etiologies can prevent/impede the development of a neurodegenerative and depressive disorder and the concomitant cognitive deficits. |
| Clioquinol | |
| Reference | Zhang et al. (2013) |
| Number of animals, gender, ages, and treatment | Adult male and female APPswe/PSEN1deltaE9 mice, 5 months of age, were assigned to 3 groups (n = 8–12/group). WT mice 5 months of age were also used. APP/PS1 mice were treated with clioquinol (CQ) 6 mg/kg p.o. once daily for 5 months. At 11 months of age, mice were anesthetized and blood collected. Euthanized mice were perfused with phosphate buffer saline followed by formalin, and the brains removed and post-fixed in formalin for histology and IHC. |
| Comparison | APPswe/PSEN1deltaE9 mice were dosed p.o with distilled water replacing the CQ stock solution. |
| Functional outcomes | Compared to untreated APP/PS1 mice, treatment with CQ significantly decreased the area fraction and the number of amyloid plaques, with the region of interest including the neocortex and hippocampus. When compared by gender, the decreases in both area fraction and plaque number in males were significantly greater than in females. In CQ treated APP/PS1 mice, conspicuous myelinopathies independent of amyloid plaques were seen in the dorsal lateral geniculate nucleus (DLG) and which consisted of numerous edematous swellings and fragmented fibers (in 11 out of 12 CQ treated mice, 92%). No myelin pathology was seen in the DLG of APP/PS1 control mice. In WT mice, relatively milder myelinopathies were seen in DLG of all of the CQ treated mice (n = 6). No myelin pathology was seen in the DLG of untreated WT mice (n = 5). Plasma levels of Aβ42 in the CQ treated APP/PS1 mice were significantly higher than those in the untreated APP/PS1 mice. Activated astrocytes were located primarily surrounding the amyloid plaques in the untreated and CQ treated APP/PS1 mice. The activated astrocytes exhibited hypertrophic processes and cell bodies and increased expression of GFAP compared with resting astrocytes. The total number of plaque-associated astrocytes was decreased in CQ treated APP/PS1 mice compared to untreated APP/PS1 mice, presumably because the area and number of amyloid plaques were reduced in CQ treated APP/PS1 mice. Activated microglia, identified by labeling with CD68, were found primarily near or surrounding amyloid plaques. There was no significant difference in the morphology of activated or resting microglia in any brain region examined between the different groups. The total number of CD68-labeled (activated) microglia was decreased in CQ treated APP/PS1 mice compared to untreated APP/PS1 in parallel with the decrease of amyloid plaques. |