Positive Effects of Probiotic Treatment on Spatial Cognitive Performance and Synaptic Plasticity in a β amyloid rat model of Alzheimer’s Disease

Positive Effects of Probiotic Treatment on Spatial Cognitive Performance and Synaptic Plasticity in a β-amyloid rat model

Amna Noor

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the presence of amyloid plaques and tau tangles in the brain. Recently, dysbiosis of gut microbiota has been implicated in etiology of various brain dysfunctions, including AD. Currently, there is no effective treatment to stop or slow down the progression of AD. As a result, therapeutic strategies focus on treating various behavioural and cognitive symptoms. The research conducted by Rezaei Asl, Sepehri and Salami (2019) addresses the lack of treatment and hypothesizes that supporting the gut microbiome with probiotics will reverse some of the negative effects of the dysbiosis. An animal model of AD was made by injecting β-amyloid intracerebroventricularly into male Wistar rats. The probiotic treatment was made up of encapsulated Lactobacillus acidophilus, Bifidobacterium bifidum, and Bifidobacterium longum. The rats were further divided into five groups: rats that received water (Con), rats that received probiotics and water (Pro + Con), rats that received the injection (Alz), rats that received injection and probiotics (Alz + Pro), and rats that underwent sham surgery (Sham). Evaluation of different behavioural and electrophysiological aspects of AD via conducting a plethora of tests confirmed restoration of synaptic plasticity (LTP), enhancement of spatial cognitive performance, and an increase in antioxidant to oxidant ratio in rats that received the probiotic concoction. Therefore, the study served as evidence for a novel treatment of AD through probiotic support of gut microbiome. Keywords: Neurodegeneration, neurodegenerative disease, Alzheimer’s disease, long term potentiation, spatial memory, animal model, gut microbiota, dysbiosis, probiotics

INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disease that was first discovered in 1906. It is now a leading cause of death and dementia in older adults. Currently, AD is characterized by the deposition of β-amyloid plaques, the formation of neurofibrillary hyperphosphorylated tau protein tangles, neuroinflammation, and progressive impairment of neuronal synapses (Selkoe 2001; Scheltens et al. 2016). However, many of its pathophysiological facets are still being examined (Viña and Sanz‐Ros 2018). There is no effective treatment to halt or slow down the progression of AD. Available options include combination therapies to manage behavioural symptoms and cognitive functions associated with AD such as memory deterioration (Selkoe 2001; Viña and Sanz‐Ros 2018). Gut microbiota refers to the population of commensal microorganisms residing in the human gastrointestinal (GI) tract. The gut microbiota is unique to every individual but has common dominant bacteria, such as Firmicutes and Bacteroidetes, whose composition changes in diseased individuals. In healthy hosts, microbiota serves to maintain a protective barrier against pathogens and lives in a state known as eubiosis whereas in diseased subjects the healthy balance of microorganisms in the GI tract is compromised and microbiota enters a state of dysbiosis (Angelucci et al. 2019; Franceschi et al. 2019). In recent years, there have been numerous reports of a gutbrain axis (GBA). Specifically, GBA is a two-way signalling pathway between the gut and the brain via the vagus nerve (Collins, Surette, and Bercik 2012). A dysbiosis in the gut has been linked to various disorders of the central nervous system, such as AD (Westfall et al. 2017). Some of these studies have established links between the gut and the hippocampus which is implicated in the formation of memories and establishment of long-term depression (LTD) and long-term potentiation (LTP) via the CA1- CA3 pathway. The gut microbiome is also subject to changes that can increase gut permeability and promote bacterial translocation due to aging, which is a risk factor for AD (Jiang et al. 2017). In particular, an increase in serum levels of inflammatory cytokines and deposition of β-amyloid plaques have been linked to the direct and indirect effects of dysbiosis in different capacities (Angelucci et al. 2019). Thus, manipulation of the microbiome serves as a good therapeutic target because if the cause-and-effect relation holds, a reverse, that is the introduction of ‘good bacteria’ into diseased individuals, should alleviate symptoms of the disease. Inspired by this connection, Rezaei Asl, Sepehri and Salami (2019) researched positive effects of a probiotic concoction composed of encapsulated Lactobacillus acidophilus, Bifidobacterium bifidum, and Bifidobacterium longum in rat models of AD made by injecting β-amyloid intracerebroventricularly. Probiotics refer to bacteria that induce favourable changes in host health (Angelucci et al. 2019). The study assessed influence of probiotic treatment on spatial learning and memory via Morris water maze test, basic synaptic transmission and LTP in the hippocampus via recording field excitatory postsynaptic potentials (fEPSPs), and change in the antioxidant to oxidant factors ratio by measuring plasma content of total antioxidant capacity (TAC) and malondialdehyde (MDA).

Figure 1. Summary of methods and major findings are summarized in the figure above. The β-amyloid injection was given intracerebroventricularly and probiotics and water were administered via an intragastric gavage. Probiotics significantly improved spatial cognitive performance in the Morris water maze test and formation of LTP in the hippocampus in (Alz + Pro) and (Pro + Con) mice while also increasing antioxidant to oxidant factors ratio which reduced apoptosis.

MAJOR RESULTS Behavioural Performances

Rezaei Asl, Sepehri and Salami (2019) used a Morris water maze test to assess task learning and formation of recent memories. The test consisted of an acquisition phase whereby animals were given time to locate the platform followed by a probe trial test whereby animals searched the maze. This was in accordance with previously established methods (Vorhees and Williams 2006). Alz rats required 50% more time than other groups to locate the platform but no difference was found in the probe trial test stage. Pro + Alz rats showed improvement and were able to locate the platform faster and finally, Pro + Con mice located the platform earliest.

Figure 2. Curves summarize the impact of probiotic administration on behavioural performances of the rat Alz, Con, and Sham models. Probiotics significantly improved the performance of rats in both Alz and Con models which is indicative of better spatial cognitive functioning and formation of memories. (Figure derived from Rezaei Asl, Sepehri and Salami (2019)).

Synaptic Transmission

Rezaei Asl, Sepehri and Salami (2019) stimulated Schaffer collaterals and measured baseline fEPSPs in the CA1 pathway to conclude the effect of probiotic administration on synaptic transmission. Application of high-frequency stimulation increased LTP in Alz + Pro as well as Pro + Con rats. Alz rats showed significantly decreased LTP compared to their counterparts. Overall, the β-amyloid injection reduced hippocampal LTP, but the effects were reversed upon administration of probiotics. This showed that Alz rat models had difficulty forming new memories and learning.

Figure 3. fEPSPs observed in the CA1 area are represented by curves in the figure. Baseline fEPSP was recorded and then 10 recordings were made and averaged every 30 minutes at time marks 30, 60, and 90 minutes. The increased amplitude of fEPSP represents enhanced LTP. (Figure derived from Rezaei Asl, Sepehri and Salami (2019)).

Plasma levels of antioxidant/ oxidant factors

Rezaei Asl, Sepehri and Salami (2019) measured the plasma levels of TAC and MDA (oxidant) to conclude the ratio of the believe that this link is via a reduction in gut inflammation. Dysbiosis is linked to inflammation of the gut which causes increased penetrance of the microbiota outside the tract leading to a compromised immune barrier. Probiotics help to reduce this inflammation by increasing the amount of short fatty acid chains that release brain-derived neurotrophic factor (BDNF) and thus reduce inflammation (Rezaei Asl, Sepehri and Salami 2019). There is a limited number of studies that examine the hippocampal dependant synaptic plasticity vis-à-vis probiotics. β-amyloid plaques have been shown to cause abnormal NMDA receptor activation, which is the main CA1 - CA3 pathway receptor. Through inference from previous literature, authors determined that β-amyloid attenuates LTP by disturbing normal amyloid plaques can stress mitochondria, which results in the generation of reactive oxidative species and free radicals. Pro

factors. This is a very reliable way of measuring oxidative stress (Katerji, Filippova, and Duerksen-Hughes 2019). Alz rat models showed increased MDA levels which were decreased by probiotic administration but remained higher than normal. They also showed decreased TAC plasma levels which were efficiently increased by probiotics. Pro + Con mice showed enhanced TAC to MDA ratio. The results indicated that Alz rat models were more prone to neuronal apoptosis and probiotics could reverse tively links to increased synaptic plasticity in a β-amyloid rat model of AD, hinting a potential therapeutic strategy. Their results are in line with current literature in that accumulation of β-amyloid impairs learning and memory functions and dysbiosis is implicated in the process. Probiotics have previously been used to show similar results (Schneider et al. 2020; Yang et al. 2020). Literature suggests that the underlying mechanism might be linked to various pathways such as immunological, hormonal, and neuronal, but since the field is relatively new, a definitive link is yet to be established. Authors

NMDA receptor and BDNF functions. However, the underlying mechanism remains undetermined (Rezaei Asl, Sepehri and Salami 2019). Lastly, the increase in antioxidant factors and a decrease in oxidant factors is also supported by the literature (Mehta et al. 2017). AD is linked to oxidative stress as βthis.

figure above. biotics increase antioxidant enzymes and reduce inflammation which reverses this phenomenon (Rezaei Asl, Sepehri and Salami 2019).

CONCLUSIONS/ DISCUSSIONS

Through this experiment, the authors were able to demonstrate that the probiotics supplement can be used to reverse behavioural and electrophysiological symptoms of AD and thus, that AD rat models that received the probiotics supplement demonstrated improved hippocampal dependant cognitive functioning and had increased plasma levels of antioxidant species reducing apoptosis (Rezaei Asl, Sepehri and Salami 2019). The research is significant because it is the first study that posiFigure 4. Major implications of the study are highlighted in the

serve as a potential therapy. This was due to their observations

CRITICAL ANALYSIS

The purpose of this study was to supplement gut microbiota with a probiotics concoction to alleviate the negative effects of dysbiosis on AD patients. Although probiotics showed great promise as a potential treatment, the authors used a combination of different bacterial genera to construct the probiotic sup-

plement and the effects cannot be attributed to a specific genus (Rezaei Asl, Sepehri and Salami 2019). A study by Magistrelli et al. in 2019 showed a similar decrease in oxidative species with the use of probiotics constructed from lactobacillus and bifidobacterium genus in patients with Parkinson’s Disease. However, they showed the most improvement in patients that received probiotics composed of L. salivarius and L. acidophilus. Bubnov et al. 2015 also report the use of Akkermansia muciniphila to maintain the integrity of the gut lining which can help reduce the negative effects of cytokines on learning and memory. As a follow-up, the authors should focus on specific genera, as well as the optimal composition of these species in the concoction, to have more confidence in conclusions. The underlying mechanisms for their conclusions were unclear due to the lack of evidence, so although they seem logical via inference, a definitive mechanism could not be determined. Furthermore, even though LTD has been linked to a decrease in memory and cognitive functioning as well, the results only account for LTP. This pattern can be seen in previous literature as well. The severity of AD, sex, and age of the patient have all been shown to be confounding variables for probiotics treatment (Agahi et al. 2018; Kim et al. 2020). The authors should have had controls for these variables as well. The rest of the methods used in the study are well-established methods used extensively in the literature (Katerji, Filippova, and Duerksen-Hughes 2019; Vorhees and Williams 2006). tioned directions, molecules secreted by gut microbiota such as vascular endothelial growth factor B (VEGF-B) and transforming growth factor alpha (TGF-α) that regulate neuroinflammation via recruitment of microglia and astrocytes should also be studied as potential mechanisms for the role of probiotics (Giau et al. 2018). Similarly, other factors that could be used in conjunction with probiotics supplement, such as exercise, should be studied, Abraham et al. in 2019 showed that probiotics and exercise therapy significantly increased water-maze performance in mice models of AD. Music has also been linked to an increase in memory retention in patients with AD (Lord and Garner 1993). These results could theoretically be used to model potential combined therapy without the need for invasive procedures. Lastly, it is also important to consider and explore the role of probiotics in reducing LTD. Successful experimentation should show the probiotics categorically reduce the induction of LTD in animal models. In conclusion, the authors provided the first proof of the positive effects of probiotics on hippocampal-dependent synaptic plasticity and several future studies can develop this further.

FUTURE DIRECTIONS

Authors of this paper were the first ones to demonstrate that probiotic supplementation has a positive influence on hippocampal-dependent synaptic plasticity in a rat model of AD and as such there was a lack of previous literature to support or contradict their results as well as to identify underlying mechanisms (Rezaei Asl, Sepehri and Salami 2019). As discussed earlier, age, sex, and severity of AD can all influence the impacts of probiotics on the gut microbiota (Agahi et al. 2018; Kim et al. 2020). The sample sizes for different groups in this study are from 7 to 10 rats which are not large enough to control for all the different variables (Rezaei Asl, Sepehri and Salami 2019). Future researchers should either include a bigger sample size with a wider range of controls in their study or study the effects of probiotics on each of the different type of patient (young female with mild AD vs old female with mild AD et cetera) to determine the efficacy of probiotics as a potential treatment. If the results show no significant differences between the groups and support the results of the study at hand, then probiotics can be used as a successful intervention or vice versa. Adding on to that, the researchers used bacteria from different species to draw their results which makes it difficult to replicate the study (Rezaei Asl, Sepehri and Salami 2019). As a follow-up to this study, the effects of individual bacterium species can be studied. Future studies should also explore multiple models of AD as it is a multifactorial disease. For example, in 2017, Mehta et al. were able to produce similar oxidative stress results in Dgalactose animal models of AD. In addition to the aforemen

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