Andrographolide attenuates short-term spatial and recognition memory impairment and neuroinflammation induced by a streptozotocin rat model of Alzheimer’s disease

doi:10.21203/rs.3.rs-1758931/v1

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Andrographolide attenuates short-term spatial and recognition memory impairment and neuroinflammation induced by a streptozotocin rat model of Alzheimer’s disease

https://doi.org/10.21203/rs.3.rs-1758931/v1

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Alzheimer’s disease (AD) is a neurodegenerative disorder clinically manifested by a gradual cognitive decline. Intracerebroventricular injection (ICV) of streptozotocin (STZ), a model of Sporadic AD (sAD), shows many aspects of sAD abnormalities (i.e., neuroinflammation, oxidative stress, protein aggregation) resulting in memory impairment. Andrographolide (ANDRO), a natural diterpene lactone, has numerous bioactivities including anti-inflammatory and antioxidant properties. Studies in rodents revealed that ANDRO has neuroprotective properties and restores cognitive impairment. In the present study, we investigated the effects of ANDRO in the ICV-STZ model relative to short-term spatial memory (Object location test (OLT) and Y maze test), short-term recognition memory (Object recognition test (ORT)), locomotor activity (Open field test (OFT)), expression of amyloid precursor protein (APP), and activation of astrocytes (glial fibrillary acidic protein (GFAP) expression) and microglia (ionized calcium-binding adapter molecule-1 (Iba-1) immunohistochemistry) in the prefrontal cortex (PFC) and hippocampus (HIP). Wistar rats were injected ICV with STZ (3 mg/kg) or vehicle and treated with ANDRO (2 mg/kg, i.p.; three times per week). After four weeks, ANDRO attenuated the impairments of the Y maze and ORT performances, and the increase of the astrocyte activation in the PFC induced by the ICV-STZ model. In addition, ANDRO decreased the number of activated microglia cells in the HIP of STZ-injected rats. The APP expression was not altered, neither by the STZ nor ANDRO. ANDRO showed a beneficial effect on memory impairment and neuroinflammation in the STZ model of AD.

Alzheimer

andrographolide

cognition

microglia

astrocyte

streptozotocin

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder and is mainly characterized by progressive and slow memory loss, especially memory related to recent events and spatial disorientation (Salardini 2019; Moura et al. 2020). Late-onset AD, or sporadic AD (sAD), is the preponderant form of the disease, despite its unclear etiology many molecular disturbances have been detected in the sAD aging brain (Correia et al. 2011). The classical positive lesions consist of accumulation of extracellular amyloid β (Aβ) and intracellular hyperphosphorylated tau (p-tau) and are accompanied by oxidative and inflammatory damage. These neuropathological aspects generate energy failure and ultimately synaptic and neuronal loss, resulting in cognitive impairment (Querfurth and LaFerla 2010; Serrano-Pozo et al. 2011). In patients with sAD, the loss of synapses in the neocortex and hippocampus (HIP) appears to be substantial to sAD’s pathology since the slow decline of the cognitive functions strongly correlates with the progression of the neuronal dysfunction in both regions (Serrano-Pozo et al. 2011; Cisternas et al. 2019).

An intracerebroventricular (ICV) injection of low, sub-diabetogenic, doses of streptozotocin (STZ) in rodents can model sAD (Bassani et al., 2017a; Grieb, 2016; Salkovic-Petrisic and Hoyer, 2007). STZ can inhibit insulin receptors, inducing an insulin-resistant brain state and brain glucose hypometabolism, abnormalities that appear early in sAD (Salkovic-Petrisic et al. 2013). In the ICV-STZ model, as in the disorder, these features develop along with an increase of free radical formation, neuroinflammation, protein aggregation, cholinergic deficits, neurodegeneration, and cognitive decline (Kamat et al. 2016). Characteristics that resemble those of the human AD brain and have been documented after the ICV administration of STZ in rats. Thus, the model is a suitable strategy to mimic human sAD conditions and it is widely used to evaluate the neuroprotective properties of various compounds (Kraska et al. 2012).

Existent AD’s therapeutic approaches are limited by the uncertainties about its etiology, there are still no disease-modifying treatments, and most agents provide only symptomatic relief from the cognitive deficit (Stella et al. 2015; Lane et al. 2018). Andrographolide (ANDRO), the main bioactive component of the medicinal plant Andrographis paniculata, is a labdane diterpenoid lactone that has multiple therapeutic uses and is largely known for its anti-inflammatory and antioxidative activities (Guan et al. 2013; Li et al. 2015; Zhang et al. 2021). It has shown beneficial effects within the central nervous system (CNS) in animal models of cerebral ischemia, traumatic brain injury, and neurodegenerative diseases (Chan et al. 2010; Tao et al. 2018; Geng et al. 2019). The treatment with ANDRO reduced cognitive impairment in different models of AD, such as APPswe/PS1ΔE9 and J20 transgenic mouse models, as well as aged Octodon degus, used as a natural model of AD (Rivera et al. 2016; Cisternas et al. 2019; Arredondo et al. 2021).

ANDRO was found to be neuroprotective and improve learning and memory by different molecular and cellular mechanisms such as reduction of Aβ and tau levels, suppression of astrocyte and microglial activation, and promotion of hippocampal neurogenesis (Serrano et al. 2014; Arredondo et al. 2021; Hossain et al. 2022). Likewise, Patel et al. (2021) have recently demonstrated that ANDRO can suppress the impairment of spatial memory caused by the ICV-STZ model in rats. ANDRO protected the hippocampus against STZ induced increase of oxidative stress, neuroinflammatory markers (interleukin-1β (IL-1β), IL-16, and tumor necrosis factor-alpha (TNF-α)), and Aβ1–42 and p-tau levels. However, the effects of prolonged ANDRO treatment on astrocyte and microglia activation and recognition memory were not yet studied in the ICV-STZ model. Therefore, we hypothesized that ANDRO would be able to preserve spatial and recognition memory of ICV-STZ injected rats by decreasing the activation of astrocytes and microglia and reducing amyloid precursor protein (APP) expression in the prefrontal cortex and hippocampus.

2.1. ANDRO partially prevented the impairment of short-term spatial memory inflicted by the ICV-STZ

Short-term spatial memory was assessed in the Y maze on day 30 after surgery, with an 1 h interval between the training and test sessions. The STZ + VEH group showed a significant decrease of the percentage of time spent in the novel arm (p < 0.05) in the Y maze (Fig. 2), compared with the SHAM + VEH group (treatment x lesion interaction [F (1, 38) = 5.881; p = 0.020]). The STZ + ANDRO group performed similarly to the SHAM + VEH group, suggesting that ANDRO prevented the memory disruption, however, the percentage of time spent in the novel arm was not increased compared to the STZ + VEH group. There were no significant differences between the groups regarding the total entries in the arms of the maze (Fig. S1a) and the total distance travelled (Fig. S1b).

2.2. ANDRO worsened the performance in the OLT and partially prevented the impairment of short-term recognition memory induced by the ICV-STZ

Short-term spatial memory and short-term recognition memory were assessed in the OLT on day 28 and in the ORT on day 29 after surgery, respectively, with an 1 h interval between the training and test sessions. In contrast to the performance in the Y maze, in OLT (Fig. 3), the STZ + VEH group did not present a decrease of the index of spatial memory. And surprisingly, the STZ + ANDRO group showed a decrease of the OLT discrimination index compared to the STZ + VEH group (p < 0.05), suggesting that ANDRO had a negative impact on short-term spatial memory in this test (treatment x lesion interaction ([F (1, 30) = 6.939; p = 0.0132]; lesion factor [F (1, 30) = 10.12; p = 0.003]), as opposed to what was observed in the Y maze.

In the ORT (Fig. 4), STZ + VEH animals presented deficits in short-term memory, as indicated by treatment x lesion interaction [F (1, 30) = 7.601; p = 0.009], with a significant decrease of the discrimination index (p < 0.05) in comparison with the SHAM + VEH group. The STZ + ANDRO group performed similarly to the SHAM + VEH group, which indicates a protective effect of ANDRO, however, the discrimination index was not superior compared to the STZ + VEH group. There were no significant differences between the groups regarding the total time exploring the objects (e) both in the OLT (Fig. S2a) and in the ORT (Fig. S2b).

2.3. ANDRO diminished locomotion frequency in the Open Field Test

Regarding the performance in the OFT (Fig. 5), assessed on day 21 after surgery, there was only a significant difference in locomotion frequency between the STZ + ANDRO and the SHAM + VEH animals as indicated by the lesion factor [F (1, 38) = 9.126; p = 0.004].

2.4. The treatment with ANDRO attenuated astrogliosis in the prefrontal cortex of ICV-STZ rats

GFAP expression in the PFC (Fig. 6a) significantly increased in the STZ + VEH group compared with the SHAM + VEH group 30 days after the surgery (p < 0.05), as showed by the post-hoc test, but not by the two-way ANOVA (treatment x lesion interaction [F (1, 12) = 4.180; p = 0.063], treatment [F (1, 12) = 0.234; p = 0.637], and lesion [F (1, 12) = 3.965; p = 0.069] factors). In the STZ + ANDRO group, GFAP expression in the PFC was comparable with SHAM + VEH animals, however there was also no significant difference in relation to the STZ + VEH group. In the hippocampus (Fig. 6b), there were no differences between the groups (treatment x lesion interaction [F (1, 12) = 1.521; p = 0.241]; treatment [F (1, 12) = 3.350; p = 0.092]; lesion [F (1, 12) = 0.824; p = 0.381]). These results suggest that the ICV-STZ model increases the GFAP expression in the PFC after 30 days, and that ANDRO attenuated the astrogliosis in this cerebral region.

2.5. Correlation between short-term memory and GFAP expression in the HIP and PFC

The Pearson’s correlation coefficients revealed negative correlations between the percentage of time spent in the novel arm in the Y maze and the expression of GFAP in the PFC (r = -0.711, p = 0.014; Fig. 7a), and between the discrimination index of the ORT and the expression of GFAP in the HIP (r = -0.653, p = 0.021; Fig. 7b). No correlation was found between the percentage of time spent in the novel arm in the Y maze and the expression of GFAP in the HIP (r = -0.137, p = 0.688; data not shown), and between the discrimination index of the ORT and the expression of GFAP in the PFC (r = -0.162, p = 0.615; data not shown).

2.6. ANDRO attenuated the increase of hippocampus Iba-1-positive cells of STZ-injected animals

The dorsal HIP was evaluated in three regions, CA1, CA3 and dentate gyrus (DG), regarding the number of Iba-1 + cells and reactive Iba-1 + cells. The two-way ANOVAs showed significant differences between the groups variances in the CA1 (treatment x lesion interaction [F (1, 12) = 5.218; p = 0.041]; treatment [F (1, 12) = 5.671; p = 0.035]) and CA3 (treatment x lesion interaction [F (1, 12) = 7.378; p = 0.019]; treatment [F (1, 12) = 4.987; p = 0.045]), but not in the DG. In relation to the number of reactive Iba-1 + cells, significant differences were observed in the CA3 (treatment x lesion interaction [F (1, 12) = 6.809; p = 0.023]) and in the DG (treatment x lesion interaction [F (1, 12) = 10.540; p = 0.007]). However, different from what we expected, the multiple comparisons tests did not reveal differences between the STZ + VEH and SHAM + VEH groups (Iba-1 + cells in CA1, p = 0.176, and CA3, p = 0.069; reactive Iba-1 + cells in CA3, p = 0.059, and DG, p = 0.051). Yet, in the comparison between the STZ-injected groups, the treatment with ANDRO reduced (p < 0.05) the Iba-1 + cells in CA1 (Fig. 8a) and CA3 (Fig. 8c), and the reactive Iba-1 + cells in CA3 (Fig. 8d) and DG (Fig. 8f). These results indicate a mild improvement by ANDRO against the increase of microglia cells and microglia reactivity.

2.7. Correlation between short-term memory and the number of Iba-1 + cells in the HIP

The Pearson’s correlation coefficients revealed negative correlations between the percentage of time spent in the novel arm in the Y maze and the number of Iba-1 + cells in the CA3 area (r = -0.530, p = 0.042; Fig. 10a), and between the percentage of time spent in the novel arm in the Y maze and the number of reactive Iba-1 + cells in the CA3 (r = -0.567, p = 0.027; Fig. 10b) and the DG (r = -0.529, p = 0.043; Fig. 10c).

2.8. ICV-STZ infusion and ANDRO treatment did not affect the APP expression in the PFC and HIP

The two-way ANOVA did not show differences between the groups in the expression of the amyloid precursor protein (Fig. 11), both in the PFC (treatment x lesion interaction [F (1, 11) = 0.008; p = 0.930]; treatment [F (1, 11) = 0.746; p = 0.406]; lesion [F (1, 11) = 0.725; p = 0.412]) and the HIP (treatment x lesion interaction [F (1, 12) = 0.122; p = 0.732]; treatment [F (1, 12) = 0.665; p = 0.430]; lesion [F (1, 12) = 0.006; p = 0.940]).

The current study investigated the effects of prolonged andrographolide treatment (2 mg/kg, 30 days) on short-term spatial and recognition memory and neuroinflammation in the ICV-STZ model of sAD in rats. It is extensively described in the literature that the ICV infusion of STZ in rodents causes impairments in cognitive tasks that rely on the HIP (Prickaerts et al. 1999; Rodrigues et al. 2010; Li et al. 2020). This feature can mimic a well-documented early symptom of AD, spatial and temporal disorientation (Tu et al. 2015). The ability of rodents to remember a location in space defined by distal visual cues, which is essential for the Y maze task, is severely disturbed by hippocampal damage (Martin and Clark 2007; Eichenbaum 2017). In the present study, as indicated by the Y maze performance, the STZ-injected animals showed impairment of short-term spatial memory. It has been previously demonstrated that ANDRO can improve the performance of rodents in spatial memory tasks (Serrano et al. 2014; Rivera et al. 2016). Here, ANDRO exhibited an ambiguous role. In the Y maze test, the results suggest a positive effect of ANDRO in short-term spatial memory. However, in the object location test, the STZ-animals treated with ANDRO had a significantly worse performance in comparison with the SHAM + VEH group, and the STZ + VEH group a similar one, suggesting a negative impact of ANDRO. The conflict between these results is unexpected since the two tasks have fundamental similarities in assessing short-term spatial memory, both are inherently not stressful and based on exploiting rodents’ innate preference for novelty (Dellu et al. 1997; Vogel-Ciernia and Wood 2014). Despite the poor performance of the STZ + ANDRO group, all the groups showed a low discrimination index in the OLT (-0.1 < d < 0.2), including the control group, which could have occurred due to performance confounds, like stress and anxiety (Vogel‐Ciernia and Wood 2014).

Along with spatial memory dysfunction, recognition memory dysfunction is proposed as an early marker of AD, been relevant for animal models of AD (Bassani et al., 2017b; Grayson et al., 2015). Therefore, we subjected the animals to the object recognition test. The ORT relies on different brain regions, including insular, perirhinal, and ventromedial prefrontal cortices. The PFC may be of particular importance for associative memory (Vann and Albasser 2011). The HIP role in object recognition varies depending on the experimental setup, in the present protocol is suggested that the hippocampus is required for encoding the spatial and temporal context of the objects (Vogel-Ciernia and Wood 2014; Grayson et al. 2015). In line with previous studies (Cisternas et al. 2019; el Sayed and Ghoneum 2020), we observed a disruption of short-term recognition memory caused by STZ, and ANDRO was able to preserve it, having a protective effect on rats’ ORT performance. Our study is the first to report an improvement in short-term recognition memory by ANDRO in the ICV-STZ model of sAD. In a recent report, it was observed that ANDRO (15, 30, and 60 mg/kg, 14 days) protected rats against STZ-induced learning and memory impairment (Patel et al. 2021). However, cognition was assessed 21 days after STZ infusion in the Morris Water Maze Test and in a modified version of the Elevated Plus Maze Test, which measures spatial long-term memory. In addition, the doses were substantially higher than the dose used in our study.

In the OFT, the diminished frequency of locomotion of the STZ + ANDRO group could suggest a negative effect of ANDRO on the spontaneous exploratory behavior of STZ-injected rats and an impairment in locomotor activity (Walsh and Cummins 1976; Choleris 2001). Yet, the lesion factor was the only factor showing significant differences, indicating that the STZ infusion was the responsible for the decrease in locomotion frequency not the ANDRO treatment. It is known that changes in these parameters could decrease the animals’ performance in subsequent cognitive tests (Moura et al. 2020). However, the exploratory behavior and/or ambulation seem to have been affected only in the OFT, the groups did not show differences in the total time spent exploring the objects in the OLT and in the ORT, in addition, there were no differences in the distance covered in the Y maze and in the number of entries in the arms. An explanation is that the OFT was performed 8 to 9 days before the other behavioral tests and the animals could have recovered the exploratory and/or locomotor capacity before the cognitive tests.

As mentioned, the hippocampus and the prefrontal cortex are critical for spatial memory and recognition memory tasks (Akirav and Maroun 2005; Barker and Warburton 2011; Eichenbaum 2017). The HIP and the PFC have distinct yet complementary roles in rats and humans and are part of a larger network of interconnected brain regions that contribute to mnemonic function (Vann and Albasser 2011). The ICV-STZ infusion can compromise the optimal functioning of these areas by increasing the expression of APP, Aβ, and tau; oxidative stress; and neuroinflammation, as expressed by microgliosis and astrogliosis (Bassani et al., 2017a; Chen et al., 2013; Hira et al., 2019; Pierzynowska et al., 2019). It is known that ANDRO can protect the PFC and the HIP from these disturbances in rodent models of AD. In APP/PS1 transgenic mice, the treatment with 2 mg/kg of ANDRO (three times per week for 4 weeks) reduced Aβ deposition, inhibited microglial activation, and decreased the secretion of proinflammatory factors in the PFC and HIP (Zhang et al. 2021). In the Octodon degus model, ANDRO (2 mg/kg, three times per week for 3 months) significantly reduced the total Aβ burden, the oxidative stress, as well as astrogliosis in the HIP (Lindsay et al. 2020). Thus, in the current study, we analyzed the effects of the ICV-STZ infusion and ANDRO in markers of neuroinflammation regarding the microglia and astroglia, and in the APP expression in the PFC and HIP.

Neuroinflammation, as reflected by astrogliosis and microglial activation, is a pathological hallmark of AD (Guo et al. 2017). The formation of amyloid plaques and neurofibrillary in the vicinity of activated microglia and astrocytes in the brain is demonstrated in animal studies and AD patients. Aβ deposition promotes the activation of glial cells, stimulating reactive gliosis pro-inflammatory signaling cascade (Serrano-Pozo et al. 2013; Heneka et al. 2015). Pro-inflammatory cytokines when chronically activated affect the processing of APP through beta-secretase, accelerating the production of Aβ. The result is a reduction of activated glial cells clearance of Aβ, leading to an increase of Aβ burden (Kaur et al. 2019). During immune responses, reactive microglia and astrocytes change their morphology and functionality. The activation of this reactive phenotype ensures the removal of injurious stimulus, protecting the brain. However, the diversification and perpetuation of activated glial cells can act as a double-edged sword that induces neurodegeneration and AD (Meraz-Ríos et al. 2013). Studies suggest a deleterious role of glial cells in the progression of multiple AD biomarkers (Furman et al. 2012).

Reactive astrocytes are characterized by increased expression of GFAP (Heneka et al. 2015). Several reports demonstrated that in the ICV-STZ model, the GFAP immunoreactivity and expression increased in the PFC and the HIP (Rai et al. 2014; Ravelli et al. 2017; Rajasekar et al. 2017; Pilipenko et al. 2019). In the present study, GFAP expression increased only in the PFC of the STZ + VEH group. ANDRO protected the PFC of the rats against astrocyte reactivity induced by STZ. In the HIP, there were no differences between groups, however, a negative correlation was identified between the performance in the ORT and the GFAP expression. Furthermore, we found a negative correlation between the performance in the Y maze and the GFAP expression in the PFC. These results corroborate the association between cognitive impairment and astrogliosis and the neuroprotective role of ANDRO in the PFC.

Distinctively from previous studies (Zappa Villar et al. 2018; Pilipenko et al. 2020) we did not observe an increase of the number of cells and reactivity of microglia in the HIP of the STZ + VEH group in relation to the SHAM + VEH group. Nevertheless, in the CA3 and the DG there were important tendencies. On the other hand, there was a significant decrease of microglia cells in CA1 and CA3, and of reactive microglial cells in CA3 and DG of the STZ + ANDRO group compared to the STZ + VEH group, agreeing with the report of Zhang et al. (2021) and reinforcing the capability of ANDRO to inhibit the hypertrophy and proliferation of microglia in regions of the HIP. In addition, we identified negative correlations between the performance in the Y maze and microgliosis in the CA3 and DG areas.

Regarding the expression of APP, no significant differences were observed between the groups both in the PFC and the HIP. There is evidence that the ICV infusion of STZ can increase APP levels. For example, Pierzynowska et al. (2019) observed augmented expression of APP in the PFC, HIP, and the rest of the brain of Wistar rats after 30 days of the STZ infusion (3 mg/kg). Retinasamy et al. (2020) observed in Sprague Dawley (SD) rats an increase of the gene expression of APP in the PFC and HIP 21 days post-surgery (STZ 3 mg/kg). However, there is also contrasting data, as in the study of Zappa Villar et al. (2018), in which the infusion of 1 and 3 mg/kg of STZ did not increase the APP expression in the HIP of SD rats after 25 days; in the work of Gupta et al. (2018), the gene expression of APP was increased by STZ infusion only in the cortex, but not in the HIP of SD rats 30 days after surgery. APP has been shown to have a key role in synapse formation and repair and is known to be upregulated during neuronal injury (Plummer et al. 2016). The increase of APP expression can be attributed to a regenerative attempt of the neurons to counteract the neuronal injury induced by STZ (Mishra et al. 2018). APP is a complex molecule that undergoes substantial posttranslational modification and at least ten different proteolytic fragments of APP have been identified. Several of these are suggested to be pathogenic, whereas others are neuroprotective (Wang et al. 2017). As the levels of the pathogenic fragments (e.g., Aβ 1–40, Aβ 1–42) were not measured in the present report it is difficult to state if the amyloidogenic pathway influenced the effects of STZ and ANDRO.

The mechanisms of andrographolide neuroprotection involve its anti-inflammatory and antioxidative activities (Lu et al. 2019). ANDRO can reduce glia-mediated oxidative damage and production of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-18, nitric oxide) by down-regulating the nuclear factor kappa B (NF-κB) pathways and activating the NF-E2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1), and the anti-inflammatory factor CD206, important molecules of the inflammatory response (Wong et al. 2016; Xu et al. 2019; Zhang et al. 2021). Furthermore, ANDRO reduces levels of tau protein, improves insulin resistance, restores glucose uptake, and inhibits glycogen synthase kinase-3 beta (GSK-3β) activity, activating the downstream Wnt/β-catenin pathway, recently implicated in regulating glucose metabolism in the brain, which is dysfunctional in AD (Rivera et al. 2016; Tan et al. 2017; Cisternas et al. 2019; Gherardelli et al. 2020). These mechanisms have not been contemplated in the current report, further studies are necessary to better understand the ANDRO role in the ICV-STZ model.

In summary, ANDRO attenuated the impairment of short-term spatial memory and short-term recognition memory induced by the ICV-STZ model of sAD in rats. Additionally, ANDRO decreased astrogliosis in the prefrontal cortex and microgliosis in the hippocampus of the animals, which could be related with the memory improvement. Further investigations of the effects of ANDRO on the ICV-STZ model are necessary to elucidate the adjacent mechanisms of neuroprotection and the treatment optimal dose.

4.1. Animals

Male Wistar rats (60–90 days old, weighing 300-350g), provided by the animal facility of the Federal University of Parana (UFPR), were housed in groups of 3–4 in polypropylene cages with wood shavings as bedding. The rats were maintained at 22 ± 2°C on a 12 h/12 h light/dark cycle (lights on at 7:00 AM); water and standard chow were available ad libitum. Before the experiment, the rats were allowed to acclimatize for 1 week to reduce environmental stress. The experiments were performed following the Brazilian Law for Animal Experimental Ethics and Care (11.794/October 8, 2008) and the guidelines of the UFPR Committee on the Care and Use of Laboratory Animals. The experimental procedures were approved by the University Ethics Board (CEUA/BIO - protocol #1315).

4.2. Experimental Design and Treatments

The rats were randomly divided into four experimental groups: SHAM + VEH, SHAM + ANDRO, STZ + VEH, STZ + ANDRO (10–11 per group). All the animals were submitted to stereotaxic surgery, the STZ groups received a single bilateral injection of STZ (N-[methylnitrosocarbamoyl]-α-D-glucosamine, Santa Cruz Biotechnology, USA) in a total dose of 3 mg/kg, dissolved in sterile saline. The sham groups received a single bilateral injection of sterile saline. The animals were treated three times per week (Monday, Wednesday, Friday) intraperitoneally (i.p.) with 2 mg/kg ANDRO (Sigma-Aldrich, USA) or its vehicle (0.9% saline and 2% dimethyl sulfoxide) for 4 weeks, starting 1h after the surgery (Varela-Nallar et al. 2015; Kanazawa et al. 2021).

After surgery, the rats were allowed to recover for three weeks, and the behavioral tests were conducted on the fourth week. The spontaneous locomotor activity was assessed in the open field test (OFT) on day 21 following surgery, and cognitive performance was evaluated in the object location test (OLT) on day 28, in the object recognition test (ORT) on day 29, and the spatial version of the Y maze on day 30 (Bassani et al., 2017b). The experimental design is presented in Fig. 1.

Posterior to the behavioral tests, a subset of animals (n = 4/group) was decapitated under chloral hydrate anesthesia (400 mg/kg, i.p.). The brains were extracted, and the whole HIP and PFC were dissected for the quantification of APP and glial fibrillary acidic protein (GFAP) by Western blot. Another subset of animals (n = 4/group) was deeply anesthetized with chloral hydrate (400 mg/kg, i.p.) and intracardially perfused for immunohistochemical evaluation of the microglia-specific marker for ionized calcium-binding adaptor molecule (Iba-1).

4.3. Stereotaxic surgery

Stereotaxic surgery was carried out as previously described (Bassani et al., 2017b; Bassani et al., 2017a; Moura et al., 2020). The animals were anesthetized with sodium thiopental (30 mg/kg, i.p.) and chloral hydrate (150 mg/kg, i.p.) and placed in a stereotaxic apparatus (David Kopf, USA). A 28- gauge stainless steel needle was lowered into each lateral ventricle (LV). The stereotaxic coordinates for ICV infusion, according to Paxinos and Watson (Paxinos and Watson 2007), were measured: anterior/posterior, − 0.8 mm from bregma; medial/lateral, ± 1.5 mm from the midline; dorsal/ventral, − 3.8 mm from the skull. An electronic pump (Insight, Ribeirão Preto, SP, Brazil) was used to control the flow of the injections at a rate of 1.0 µl/min over 4.5 min. The lesioned group received bilateral ICV injections of STZ (3 mg/kg total dose) dissolved in sterile 0.9% saline (4.5 µl per injection site). Sham surgery followed the same procedure, but sterile saline was injected instead of STZ. After surgery, all the rats were allowed to recover from anesthesia for 2–4 h in a heated and well-ventilated room. Food and water were placed inside the cage for 10–15 days so that the animals could easily access it without physical trauma caused by head surgery.

4.4. Open Field Test

The OFT was performed 21 days after surgery. The open field apparatus was placed in a moderately lit room and consisted of a circular arena (97 cm diameter, 42 cm height) which was divided into three concentric circles and subdivided into 19 quadrants. A video camera was placed right above the arena to record the animal's behavior for posterior analysis. Individually the animals were placed in the center of the apparatus and allowed to freely explore it for 5 min. The total number of crossings from one quadrant to another was measured. A crossing was considered only when the animal entered another quadrant with its four paws. The apparatus was cleaned with a 20% ethanol-water solution between each test to eliminate possible odors left by other rats.

4.5. Object location test (OLT) and object recognition test (ORT)

The OLT and ORT were performed between days 27 and 29 following surgery to evaluate short-term spatial and short-term recognition memory, respectively. Both tests took place in a squared arena, 100 cm × 100 cm × 40 cm, made of wood and painted black. It was placed in a moderately lit room. A video camera was positioned over the arena, and the animals’ behavior was recorded for later evaluation.

The first procedure consisted of the habituation of the animals in the box. Each animal was placed in the empty apparatus for 5 min for free exploration. Twenty-four hours later, a novel habituation session of 5 min was performed. After a 1 h delay, during the training session, two identical objects were placed in the apparatus in a symmetrical position about 10 cm away from the wall. The animals were allowed to explore them freely for 5 min and were then returned to their home cages. In the OLT (which was performed on day 28), after a 1 h delay, during the test session, each rat was put back into the box with one of the objects displaced 15 cm away from the original position (novel position); animals were allowed to freely explore the objects for 3 min. In the ORT (which was performed on day 29), a new training session was done with the two identical objects placed in a symmetrical position about 10 cm away from the wall, and the animals were allowed to explore them freely for 5 min and were then returned to their home cages. After a 1 h interval, the rats were put back into the arena for the test session; but now with two dissimilar objects, a familiar one (the sample) and a new one, the animals were allowed to freely explore the objects for 3 min.

The objects were made of plastic or ceramic. To avoid an olfactory bias, before each trial, the objects were cleaned with a 20% ethanol solution. All objects and locations were balanced to reduce potential biases due to preferences for particular locations or objects. A rat could not displace the objects and the subjects were always placed into the box facing the same wall. Exploration was defined as sniffing at no more than 2 cm or touching the objects with the nose and/or forepaws. Sitting on or turning around the objects was not considered exploratory behavior. The animals that spent less than 10 s exploring the objects were excluded from the test.

The measures for both the OLT and ORT were the time spent by the rats exploring each object during the test session. The time spent exploring the familiar and the new object (ORT)/displaced object (OLT) was represented by 'a' and 'b', respectively. The following variables were calculated: e = a + b, and d = (b − a)/e. The 'e' variable is a measure of the total exploration time of both objects during the test session. 'd' is considered a discrimination index between the new and the familiar objects/locations, and a relative measure of discrimination that corrects for the exploratory activity (e) (de Bruin et al., 2011).

4.6. Spatial Version of Y maze

A symmetrical Y maze was constructed of wood and painted black. It consisted of three arms. The arms (50-cm in length, 27- cm in height, 12-cm in width) were arranged at a 120° angle relative to each other. The test consisted of a training session and test session that were performed at 1-h intervals. In the training session, one arm was made inaccessible by a removable blockade that was placed in front of it. A rat was placed in one of the other arms (i.e., "start arm"). The start arm was randomized between groups. The animals were then allowed to explore these two arms for 5 min. The rat was then removed from the arena and returned to its home cage. After a 1-h interval, in the test session, the rat was returned to its corresponding start arm, but the blockade that prevented access to the third arm (i.e., "novel arm") was removed, thus providing access to all three arms. The rat was allowed to explore the three arms for 3 min. The test session was video recorded for subsequent analysis. Short-term spatial memory was assessed as the percentage of time spent on the novel arm, which had to be significantly greater than 33.3% of the total time on the maze (corrected for the latency to move from the start arm to another arm and the time spent in the center of the maze). An arm entry was considered when both hind paws were placed completely inside an arm. The maze was cleaned between sessions with a 10% ethanol solution to reduce olfactory bias (Kraeuter et al. 2019).

4.7. Quantification by Western Blot

To investigate the effects of ANDRO on the astroglia in the HIP and PFC of ICV-STZ rats, we measured the level of GFAP, the commonly used marker for astrocytes, via western blot. The samples were homogenized and sonicated in lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris-HCl, pH 8.0, and protease inhibitors). After centrifugation at 20,000 rpm and 4°C for 20 min, the supernatant was collected, and the protein concentration was determined by the Bradford method (Bio-Rad, Germany). The supernatants were boiled with Laemmli buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris-HC with adjust final pH of buffer to 6.3) for 10 min at 95ºC. After reduction, the samples were subjected to electrophoresis and transferred to nitrocellulose membranes (Bio-rad, 0.45 µm). The membranes were blocked with 5% nonfat milk in TBS-Tween 20 solution, and then incubated overnight at 4ºC with mouse monoclonal anti-β-amyloid antibody (1:750; sc-28365, Santa Cruz Biotechnology, Santa Cruz, USA), rabbit polyclonal anti-GFAP antibody (1:1000; ab7260, Abcam), rabbit polyclonal anti-β-actin (1:500; sc-130656, Santa Cruz Biotechnology, Santa Cruz, USA), or mouse monoclonal anti-GAPDH antibody (1:5000; sc-32233, Santa Cruz Biotechnology, Santa Cruz, USA). After incubation with the primary antibody, the membranes were extensively washed with TBS-T and incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (Sigma, USA) in a blocking solution for 1h at room temperature. The membranes were then washed, and the visualization was performed with Pierce ECL kit (Thermo scientific) chemiluminescence substrate on an Amersham™ Imager 600 detection system (GE Healthcare, São Paulo, SP, Brazil). Protein levels were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

4.8. Immunohistochemistry

The effects of ANDRO on the microglia in the HIP and PFC of ICV-STZ rats were explored, and immunostaining to identify microglia was carried out using Iba-1 primary antibody. Brain sample processing and immunohistochemistry reactions were developed as described by Moura et al. (2020). The animals were deeply anesthetized and then euthanized with intracardiac perfusion of saline phosphate-buffered solution (pH 7.4; 1,000 mL/kg; 4ºC), followed by 4% paraformaldehyde in phosphate-buffered solution (pH 7.4; 1,000 mL/kg, 4ºC). Then the brains were removed and placed in 30% sucrose solution for 4 days for tissue cryoprotection. After that, the tissue was protected with plastic, quickly frozen in liquid nitrogen, and stored in a freezer (–80ºC) for subsequent sectioning. The frozen tissue was cut into 30 µm semi-serial coronal sections along the dorsal hippocampus at a temperature of − 25ºC using a cryostat (Leica Biosystems, Germany). The dorsal hippocampus was collected, and the sections were placed serially in ten well-plates using − 2.56 to − 4.52mm stereotaxic coordinates to the bregma, totaling six to eight 30µm cuts per well-plate. Brain sections were stored at − 20ºC in a cryoprotectant solution containing 30% ethylene glycol and 15% sucrose in a 0.05 M phosphate-buffered solution for subsequent processing.

The free-floating method was used for immunohistochemical reactions. The brain sections were initially washed with buffer A (0.1 M PBS, pH 7.4 with 0.5% Triton X-100) and incubated in 0.1M citrate buffer, pH 6.0 in a water bath at 50ºC for 30 min. The steps described above characterize an antigen retrieval step for this marker. After that, the sections were cooled to room temperature, washed with buffer A, incubated with 0.5% H2O2 solution in 0.1M PBS for 30 min at room temperature and protected from light, washed again with buffer A and incubated with 2% bovine serum albumin (BSA) in buffer A (blocking buffer) at room temperature for 1 h. Subsequently, the plates containing the sections were incubated overnight at 4ºC with the primary antibody diluted in anti-IBA1 goat polyclonal blocking buffer (1:500, Abcam, Cambridge, USA).

The following day the sections were washed in buffer A and incubated with biotinylated secondary antibody at 4ºC for 2 h, washed again in buffer A and incubated with ABC reagent (Vectastain Elite ABC Kit, Vector Laboratories, USA) in 0.1M PBS in room temperature for 2 h. After being washed with 0.1MPBS, the reaction was revealed by incubating the sections with 3,3'-diaminobenzidine (DAB, Vector Laboratories, USA) in 0.1MPBS at room temperature for 7 min. The sections were washed in 0.1M PBS, mounted on gelatinized slides, and air-dried. The brain slices were mounted on microscope slides, dehydrated in solutions of ascending ethanol concentrations, cleared in xylene, and coverslipped.

The areas of the hippocampus (CA1, CA3, DG) were photographed using a BX50 optical microscope (Olympus Optical, USA) at 200× magnification. Microglial cells were identified as Iba-1-positive (Iba1+) cells and manually counted using the Cell Counter of the ImageJ software. Iba1 + cells were morphologically classified as Types I, II, III, IV, and V as previously described by as (Diz-Chaves et al. 2012). Types I, II, and III were categorized as non-reactive glia, whereas Types IV and V were taken as reactive glia (Zappa Villar et al. 2018). Results were expressed as a percentage of the number of sham Iba-1 + cells and sham reactive Iba-1 + cells.

4.9. Statistical Analysis

The data are presented as mean ± standard error of the mean (SEM) and analyzed using a two-way analysis of variance (ANOVA) (factor lesion: STZ or vehicle injection; factor treatment: ANDRO or vehicle treatment). Pearson’s correlation coefficient was used to evaluate the degree of association between variables. The level of significance was p < 0.05. The analysis was performed with the software GraphPad Prism 8.0 (GraphPad Software, Inc) and STATISTICA (data analysis software system) version 12 (StatSoft, Inc).

Acknowledgments We thank Professor Marcelo M.S. Lima (Department of Physiology, Federal University of Parana) for allowing us to conduct part of the experiments in the facilities of the Laboratory of Neurophysiology, and Professor Silvio M. Zanata (Department of Basic Pathology, Federal University of Parana) for the support.

CRediT authorship contribution statement Leonardo C. Souza: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization. Marcos K. Andrade: Formal analysis, Investigation, Data Curation. Evellyn M. Azevedo: Formal analysis, Investigation, Data Curation. Daniele C. Ramos: Investigation, Writing - Review & Editing. Ellen L. Bail: Investigation. Maria A. B. F. Vital: Conceptualization, Methodology, Resources, Writing - Review & Editing, Visualization, Supervision, Project administration, Funding acquisition.

Funding This work was supported by grants from Coordination for the Improvement of Higher Education Personnel (CAPES) and Araucaria Foundation to Support Scientific and Technological Development of the State of Paraná (No. 88882.168580/2018-01), which had no further role in the study design, collection, analysis, and interpretation of the data, writing the report, and decision to submit the paper for publication. MABFV is a recipient of a National Council for Scientific and Technological Development (CNPq) fellowship (No. 309567/2019-0).

Ethics Approval The experiments were performed following the Brazilian Law for Animal Experimental Ethics and Care (11.794/October 8, 2008) and the guidelines of the UFPR Committee on the Care and Use of Laboratory Animals.

Conflict of Interest The authors report no conflicts of interest.

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Andrographolide attenuates short-term spatial and recognition memory impairment and neuroinflammation induced by a streptozotocin rat model of Alzheimer’s disease

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Abstract

Figures

1. Introduction

2. Results

2.1. ANDRO partially prevented the impairment of short-term spatial memory inflicted by the ICV-STZ

2.3. ANDRO diminished locomotion frequency in the Open Field Test

2.4. The treatment with ANDRO attenuated astrogliosis in the prefrontal cortex of ICV-STZ rats

2.5. Correlation between short-term memory and GFAP expression in the HIP and PFC

2.6. ANDRO attenuated the increase of hippocampus Iba-1-positive cells of STZ-injected animals

2.7. Correlation between short-term memory and the number of Iba-1 + cells in the HIP

3. Discussion

4. Material And Methods

4.1. Animals

4.2. Experimental Design and Treatments

4.3. Stereotaxic surgery

4.4. Open Field Test

4.5. Object location test (OLT) and object recognition test (ORT)

4.6. Spatial Version of Y maze

4.7. Quantification by Western Blot

4.8. Immunohistochemistry

4.9. Statistical Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1