Supplementation of Lactobacillus Probiotic Strains Supports Gut- Brain-Axis and Defends Autistic Deficits Occurred by Valproic Acid-Induced Prenatal Model of Autism

Autism is a chronic neurodevelopmental disorder manifested by complex neurobehavioral conditions.1 Such as deficient in communication, interaction with people, repetitive movements, behave alone, deficient in motor functions, poor eye contact, etc.2 Autism occurs 4-5 times more often in boys than girls.3 Possible causes of autism are genetic, environmental factors, maternal toxicity and nutritional deficiency.4


INTRODUCTION
Autism is a chronic neurodevelopmental disorder manifested by complex neurobehavioral conditions. 1 Such as deficient in communication, interaction with people, repetitive movements, behave alone, deficient in motor functions, poor eye contact, etc. 2 Autism occurs 4-5 times more often in boys than girls. 3 Possible causes of autism are genetic, environmental factors, maternal toxicity and nutritional deficiency. 4 Gut and brain have bidirectional communication through the enteric nervous system (ENS) and vagus nerves. Such interactions can be mediated by the metabolites produced by gut bacteria called short chain fatty acids (SCFA). 5,6 The human gut is inhabited by more than a trillion (approx. 1kg) of gut bacteria (Probiotics) in the right colon. 7 Gut bacteria have symbiotic co-evolution with the host gut, in return, it improves gut health and functions by (immune support, reduces inflammatory mediators (IL-6, TNF-alpha, etc.), reduces harmful bacteria, production of nutrients and their absorption. 8,9 Gastrointestinal (GI) symptoms such as abdominal pain, constipation, diarrhea, are common comorbidities with autism. 10 Evidences related that GI symptoms are because of dysbiosis of the gut. 11 At present, there are no effective therapies for autism. A risk-free and effective treatments are necessary for autism. Modulation of the gut microbiota is a potential therapy in people with autism. Prebiotics and probiotics and fecal microbiota transplantation are the various therapeutic opportunities . 7,11 .
Probiotics, such as the lactic acid-producing bacteria belonging to Lactobacilli, Bifidobacteria and Saccharomycetes, are beneficial to the host when we provided in adequate quantities. 12,13 Many studies have shown that probiotics can prevent and treat a variety of diseases, such as obesity, depression, colorectal cancer and Crohn's disease, in animal models and humans. 14,15 Valproic acid (VPA) is an antiepileptic drug used to treat simple or complex seizures. 16 Usage of VPA in pregnant women with epilepsy lead risk effects in the fetus such as spina bifida, cleft palate, limb defects and cardiac malformations. 17 VPA is a potent teratogen, exposure of VPA during the prenatal or postnatal period can serve as triggering factors of oxidative stress which disrupt the neuron growth and development. 18 With the VPA induced autism model is a valuable tool to investigate the neurobiology, autistic behavior and to screen the several novel therapeutic agents. 19 Rats were subjected to behavioral testing to assess Negative geotaxis, Eye-opening, Swimming performance, T-maze, Morris water maze, and Social interaction studies were performed on various postnatal days up to PND 50. All behavioral studies we performed at the Neuroscience lab, School of Pharmacy, Anurag University through a video tracking system provided by VJ Instruments.

Negative geotaxis
Negative geotropism was tested on postnatal days 7 -10 by placing the mouse face down along a 45° incline in a temperature-controlled environment. Latency to turn 180° such that the head was facing upward along the incline was recorded with a maximum of 30 sec for each trial. 19

Eye opening
Eye-opening was observed daily every day after birth PND 12-16. Eye openings were recorded and rated as follows: 0-Both eyes are closed, 1-one eye-opening, 2-Both eyes are opened. 19 Swimming performance An aquarium filled with water (28-29°C) was used for swimming tests on PNDs 22, 24 and 26. Each animal was put at the center of the aquarium and was observed for 5-10s. The swimming performance was evaluated according to the position of nose and head (angle) on the surface of the water. The angle of swimming was rated as follows: 0head and nose below the surface; 1-nose below the surface; 2-nose and top of head at or above the surface but ears still below the surface; 3-the same as two except that water line was at mid-ear level; and 4-the same as three except that water line was at the bottom of ears. Thereafter, the test pups were dried and returned to the home cages. Swimming is a measure of motor development and the integration of a coordinated series of reflex responses. 19

T-maze test
The T-maze is a spontaneous alteration test, which was performed on PND 29-31, it is used to evaluate the repetitive/restricted behavior. Five sessions were performed for each rat. For each session, the first choice of the rat in the free choice arms was evaluated, i.e., whether the rat first entered the left or right arms. The parameter analyzed was the percentage of alterations between the left and right arms, which was always assessed in relation to the arm visited in the previous session. This model is based on the natural proclivity of rats to alternate between the visited goal-arms in each trail over a series of successive trails. Thus, a higher percentage of alteration between the arms was considered normal rat behavior, whereas fewer alterations indicated cognitive inflexibility and repeated behavior. For statistical analysis, these data into scores: 0= no alterations (repeatedly visiting the same arm for all five sessions), 1=one alterations, 2= two alterations, 3= three alterations, and 4= four alterations. 21

Morris water maze
Memory was evaluated by Morris water maze, autistic individuals have deficits in memory, and this test was performed on PND 48-50. The aquarium is made up of 4 Quadrants indicated by different colours for visual clues, it was filled with normal water and covered with milk, and a removable hidden platform was placed at one quadrant. Each animal was placed and the escape latency period was recorded, repeat the same for next day PND 49. On PND 50 platform was removed and identify escape latency period. 19

Social interaction
Social Interaction was performed on PND 36-40, prior to the experimentation animals are separated and housed individually overnight before the experiment. Two animals from the same group, but different litters were placed into a circular cage provided by ambient light and temperature for 20 min. frequency of the following parameters is assessed: Allogroming, Anogenital Inspections, Pinning's, Play Behavior, Social exploration. 17,21 Stereotype/ repetitive behavior The exploratory behavior of the rat was evaluated by the open-field habituation task method. A rat was placed in a 40 cm×50 cm×60 cm open field whose brown linoleum floor was divided into 12 equal squares by white lines and left to explore it freely for 5 minutes on PND 46-48. The number of line crossings and head dipping was counted. 17

BIOCHEMICAL PARAMETERS
Rat brains were isolated and washed with ice-cold 0.1M phosphate buffer pH-7.4 to remove the blood. Homogenize the brain with 0.1M phosphate buffer saline solution. Then homogenate and the resultant supernatant were used for further biochemical estimations, such as AchE, 5-HT, BDNF, and antioxidants.

Estimation of acetylcholinesterase (AchE) activity
The AchE activity was measured in brain tissue by the reaction of thiocholine with dithiobisnitrobenzoate ions. The rate of formation of thiocholine from acetylcholine iodide in the presence of brain cholinesterase was measured using a spectrophotometer (Shimadzu 1800) at a wavelength of 412 nm. 22,23 Estimation of BDNF, 5-HT, IL-6 and TNF-α The biochemical parameters was measured in the blood and brain sample by ELISA method. To the pre-coated micro ELISA plate samples and standards were added. Then addition of further substrates to conjugate and getting of specific color. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Rat BDNF, 5-HT, IL-6 and TNF-α. Calculate the concentration of Rat BDNF, 5-HT, IL-6 and TNF-α in the samples by comparing the OD of the samples to the standard curve. [24][25][26][27] HISTOPATHOLOGY On PND 50 rats was sacrificed, isolated brains were placed immediately in 10% neutral formalin solution. Processed and embedded in paraffin. Sagittal sections of the cerebellum (5µm thick) were stained with hematoxylin and eosin (H&E) and analyzed using a light microscope for changes in the cerebellum.

STATISTICAL ANALYSIS
All data are presented as Mean ±S.E.M. The significance of difference among the groups was assessed using a one-way analysis of variance (ANOVA) followed by Kolmogorov-Smirnov test, Bonferroni's Multiple Comparison Test using GraphPad Prism 5 software and (p< 0.05) was considered significant.

Effect of Lactobacillus strains on negative geotaxis
There was a significant increase in time taken to re-orient along the inclined plane seen in autistic group on PND 9-21, when compared to vehicle group (p<0.001). Treatment with Lactobacillus strains (L. Plantarum, L. Casei, L. Acidophilus, L. Bulgaricus) finally decreased the time taken to re-orient (p<0.001) with the autistic group, the results are shown in Tables 1 & 2.

Effect of Lactobacillus strains on swimming performance
There was decreased swimming performance in the autistic group compared with the vehicle group (p<0.001).

Effect of Lactobacillus strains on T-maze
There was a low alteration score reported in the autistic group by the T-maze test on PND 29-31 when compared with the vehicle group (p<0.001). Treatment with Lactobacillus strains (L. Plantarum, L. Casei, L. Acidophilus, L. Bulgaricus) showed that alteration score was significantly improved when compared with an autistic group (p<0.05) the results are shown in Figures 7-9.

Effect of Lactobacillus strains on Morris water maze
There was poor cognition seen in the autistic group with the identification of hidden platform on PND 48-50 when compared with the vehicle group (p<0.001). Treatment with Lactobacillus strains (L. Plantarum, L. Casei, L. Acidophilus, L. Bulgaricus) showed that latency to identify the hidden platform was significantly increased than autistic subjects ( p<0.001) the results are shown in Table 3.

Effect of Lactobacillus strains on Social interaction
In the autistic group, there was a lower level of social interaction seen when compared to the vehicle group (p<0.001) on PND 36-   Table 4.  Table 5.

Estimation of IL-6 & TNF-α
Prenatal induction of autism resulted in significant increase in IL-6 & TNF-α levels (p<0.001) when compared with the vehicle group. Lactobacillus strains treatment significantly attenuated the increased IL-6 & TNF-α levels (p<0.001) when compared with the autistic group. The results are shown in Table 6.

Estimation of BDNF
Significantly elevated levels of BDNF were seen in autistic group (p<0.001) when compared with the vehicle group. Lactobacillus strains treatment decreased the BDNF levels (p<0.001) when compared with the autistic group. The results are shown in Table 7.

Estimation of 5HT
Autistic rats have shown hypersertonomia when compared with the vehicle group (p<0.001). Lactobacillus strains treatment significantly decreased serotonin levels activity (p<0.001) when compared with the autistic group. The results are shown in Table 7.

Effect of Lactobacillus strains on AChE levels
In the autistic group increased acetylcholinesterase activity seen when compared with the vehicle group (p<0.001). Lactobacillus strains treatment significantly attenuated the increased enzyme activity (p<0.001) when compared with the autistic group. The results are shown in Table 7.

DISCUSSION
According to the results of this study, prenatal model of autism resulted in delayed delivery, low birth weight of pups and behavioral anomalies. The lactobacillus strains treatment ameliorated the autistic like behaviors. The offspring who exposed to VPA in the uterus showed impairment in social interaction, memory and other behavioral tests.
Negative geotaxis is an assessment parameter to motor performance of cerebellum. In autistic group increase time to reorient on the inclined plane. 19,28,29 . Reorientation time was decreased by lactobacilli. There was a delay in Eye-openings in autistic rats 19 on PND 12-16, which were become normal in the lactobacillus treatment.
Swimming performance was evaluated to measure motor development. Lower swimming activity is seen in autistic group 19,30. . Swimming rate was improved in lactobacillus treatment from PND 24 onwards. Hippocampus essential in spatial memory and learning behavior. In the T-maze test lack of spontaneous alterations is observed in the autistic group 1 . With the lactobacillus treatment it re-established the spontaneous alteration.
Morris water maze test an important tool to assess the cognitive ability. Autistic group increased latency to swim and identification of hidden platform is seen 19 . With lactobacillus treatment improved navigation skills and memory performance contributing its impact on Gut-Brain communication.
One of the important confirmation test for autism is social interaction. This test is used to assess the different patterns of behavior, communication and play behavior. With the results social interaction studies in rats were similar to autistics. In our study we identified impairments of play behavior, pinning, social explorations and repetitive behavior become normal with the autistic subjects. 1,19,30 .
Cytokines including TNF-α, IFN-γ, IL-1β, and IL-6, 12 have been reported to be elevated in the blood of autistic subjects 31 . In the CNS IL-6 can trigger inflammation and demyelination 32 . Our study evaluated the levels of TNF-α in serum, IL-6 in the cerebellum. In autistic animals IL-6 and TNF-α levels were increased with the lactobacillus treatment IL-6 and TNF-α activity become normal by their immune supportive mechanisms 33,34 .
BDNF is a neurotrophic factor (NF) that supports the growth, survival of developing and mature neurons of cholinergic, dopaminergic, and serotonergic neurons 35 . NF is more active in the hippocampus, cortex, and forebrain involved in learning, memory and higher thinking. In our study increased BDNF levels seen in serum of autistic subjects 36,37 . Treatment with lactobacillus strains BDNF activity normal with that of vehicle group.
Hyperseretonomia is seen in 25-50% autistic cases, hyperserotonemia is considered to be one of the most-well replicated findings in neurobiology. 38 5-HT or Serotonin is originated in the gut has a pivotal role in Gut-Brain communication. Some biological factors that cause the platelet hyperserotonemia may play a role in the early development of the autistic brain. 39,40 in our study we examined hyperseretonomia 41,42 in autistic group treatment with lactobacillus strains attenuated the hyperseretonomia condition, improved social behavior and communication by attributing to its effect on balancing 5-HT release from gut.
Acetylcholine is involved in the learning and memory, acetylcholine (ACh) in the synaptic cleft by hydrolysed by acetylcholinesterase (AChE). 43 A good level of acetylcholine has a regulating role in attention, cognition, social interactions and stereotypical behaviors. [44][45][46][47] in results due to excessive oxidative stress by VPA up-regulation of acetylcholinesterase seen in the autistic group. 48 Treatment with lactobacillus strains significantly decreased AchE levels by attenuating the enzyme activity.
Histopathology results of cerebellum reveals damage and apoptosis of molecular layer, granular layer seen in the autistic group. 28 Treatment with lactobacillus strains reveals mild degeneration, few necrotic cells reported in L. Plantarum and L. Bulagricus. Cerebellum become normal is seen L. Acidophilus and L. Casei and Multilactobacillus strain treatment groups. The recovery of cerebellum providing an important link connecting probiotics role in Gut and Brain communication, immune and brain health support.

CONCLUSION
In conclusion, by comparing all behavioral and biochemical results and histopathology of the cerebellum, It can be emphasized that daily supplementation with Lactobacillus strains provides a healthy gut by the enrichment of microbial diversity and re-colonization of useful bacteria that can be able to alter the brain functionality by Gut-Brain-Axis. Among we studied for four lactobacillus strains, L. Acidophilus and L. Casei given potential outcomes in recovering autism. Multilactobacillus combination is more approachable therapy, it can advances the benefit of probiotic treatment. According to our results, daily supplementation of Lactobacillus is very effective in the recovery of autism associated Dysbiosis. The present findings of this research work contributing to an important understanding on influence of gut microbiota on brain health. Further our studies will continue on selected probiotic strains of Bifidobacterium, Saccharomyces, Streptococcus and their combination with potent natural drugs.

HIGHLIGHTS
• VPA at a dose of 400mg/kg, i.p. on ED 12 to the pregnant rats has induced autism in born rats.
• Lactobacillus strains (L. Plantarum, L. Casei, L. Acidophilus, L. Bulgaricus) with good potential health benefits are selected for the study.
• Daily supplementation with Lactobacillus strains NLT 1 Billion CFU/ml ameliorated the Behavioral & Biochemical deficits.
• Lactobacillus has proven its therapeutic efficacy in autism-associated dysbiosis.