Animals
Forty young male western Albino rats, 3-4 weeks in age, weighing about 60-70 g, obtained from the experimental surgery and animal laboratory, were enrolled in the present study. The animals were randomly assigned into six experimental groups; each of them included eight rats. The animals in the control group (I) were orally given phosphate-buffered saline. The animals in the PPA-treated groups (II, III) were orally administered a neurotoxic dose of PPA (250 mg/kg body weight/day) for three days. Group II was killed after three days while group III stay alive to be killed with other groups (El-Ansary et al., 2012). The rats in the three therapeutic groups (IV, V, VI) received the same doses of PPA followed by 0.2 g/kg body weight of probiotic (ProtexinR), healthy bacteria Bifidobacterium infantis, and healthy bacteria Lactobacillus bulgaricus respectively for three weeks. ProtexinR (Somerset, UK) is a mixture of some healthy bacteria like Bifidobacterium infantis, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus rhamnosus, Streptococcus thermophiles with the concentration of 1 billion CFU per gram.
The rats were placed at 22 ± 1 °C with ad libitum access to water and standard chaw, and quantitative stool cultures were carried out. The experiment protocol was accordance with the ethical standards of the ethics committee responsible for animal experimentation at King Saud University, Riyadh, and was approved according to the Helsinki Declaration of 1975, as revised in 2008 (http://www.wma.net/en/20activities/10ethics/10helsinki/). (IRB NO.: KSU-SE-19-131).
Preparation of brain tissue homogenates
By the end of the feeding trials, deeply anesthetized (by using Ketamine/Xylazine + D.W (91, respectively 9 mg/kgbw, I.P.) rats were decapitated. The brain tissues were taken from the rats in the six groups and dissected into small pieces, homogenized in bidistilled water (1:10, w/v), and stored at -30 °C until further use.
Biochemical analyses
The quantitative determination of all tests was measured according to the manufacturer's instructions using ELISA kits from MyBioSource (San Diego, USA). Gamma-Aminobutyric Acid Receptor Subunit Alpha-1(GABRA1) (Cat No: MBS9342109), GABA (Cat No. MBS269152), glutamate (Cat No. MBS 269969), glutamine (Cat No: MBS755884) and Glutathione S-Transferase (Cat No: MBS564158). The parameters were measured in brain tissue homogenate from all the experimental animal groups, using the enzyme-linked immunosorbent assay (ELISA) technique. The applied assays were based on the method of competitive binding enzyme immunoassay technique. Descriptions of kits-assays are described below.
Measurement of Gamma-Aminobutyric Acid Receptor Subunit Alpha-1 (GABRA1) Concentration:
This assay is based on a Quantitative Sandwich ELISA kit. from MyBioSource, USA (Cat No: MBS9342109). In the kit’s plate 50μl of, Standard was added to each Standard well, 50μl sample was added to each sample well, and to each Blank/Control well Sample Diluent 50μl was added. This kit, based on the competitive enzyme immunoassay technique, uses HRP-conjugate reagent added to each well followed by incubation for 60 minutes at 37°C after being covered with a Closure Plate Membrane then 4 the plate was washed four times until excess conjugate and unbound standard, or sample was removed from the plate. Next Chromogen Solution A 50μl and Chromogen Solution B 50μl were added to each well successively to be incubated for 15 minutes at 37°C away from light to. 50μl Stop Solution was added to each well. The color in the wells was changed from blue to yellow. Finally, the Optical Density (O.D.) at 450 nm was measured by using an ELISA reader within 15 minutes after adding Stop Solution.
Measurement of Glutamate (Glu) Concentration:
Glutamate was analyzed using an ELISA diagnostic kit from MyBioSource (San Diego, CA, USA) (Cat No. MBS269969). The principle of the kit was a Double Antibody Sandwich based on characteristics of the tested antigen with more than two valances that can identify coated antibody and detection antibody at the same time. First, the extraction samples and the standards were added together with the diluent in the extraction plate. Next derivatization with sodium hydroxide, the equalizing reagent, and D reagent in a reaction plate, covered, and shaking for 2 h, followed by addition of the Q Buffer into all wells. In the last step, the extracted samples and standard compete with glutamate antiserum for a specific number of binding sites on the glutamate microtiter strips, a mechanism lasting for 15–20 h at 2–8°C. Free antigen and free antigen-antiserum complexes are then removed by three times wash. After that, the anti-rabbit IgG-peroxidase conjugate is added, incubated for 30 min on a shaker, and aspirated by washing.
Tetramethyl benzidine (TMB), as the enzyme-substrate, was then added to detect the antibody bound to the solid phase. The absorbance of the solution in the wells is read with the use of a microplate reader at 450 nm, and the concentration of unknown titers was calculated using the standard curve.
Measurement of Gamma-Aminobutyric Acid (GABA)
Gamma-aminobutyric acid was analyzed using an ELISA kit from MyBioSource (San Diego, CA, USA) (Cat No. MBS269152). The kit has a microtiter plate that is pre-coated with an antibody specific to GABA. During the assay, the GABA in the standard or sample competes with a fixed quantity of biotin-labeled GABA for sites on the pre-coated monoclonal antibody. After washing the excess conjugate and unbound standard or sample from the plate, avidin conjugated to horseradish per- oxidase (HRP) was added. TMB as an enzyme-substrate was used to detect the antibody bound to the solid phase, the developed color was read at 450 nm, and the concentration of GABA was measured using the standard curve.
Measurement of Glutamine Concentration:
For the measurement of glutamine quantity in rats’ brain tissue, an ELISA kit from MyBioSource (San Diego, CA, USA) (Cat No: MBS755884) was employed. This kit, based on the competitive enzyme immunoassay technique, uses a GLN-HRP conjugate and a monoclonal anti-glutamine. On the pre-coated plate, the assay sample and the buffer are incubated for 1 h together with GLN-HRP conjugate, then decanted, and then five times washed. Then, the wells are incubated with TMB as a HRP enzyme substrate. The intensity of the yellow color that appeared after adding the stop reagent was spectrophotometrically measured at 450 nm in a microplate reader.
Measurement of Lipid Peroxidation Concentration:
The sensitivity of measuring Thiobarbituric Acid Reactive Substances (TBARS) has made this assay the method of choice for screening and monitoring lipid peroxidation major indicator of oxidative stress. The assay has provided important information regarding free radical activity in disease states and is used for measurement of antioxidant activity, it remains the most widely employed assay used to determine lipid peroxidation.
Firstly, labeled each disposable glass test tube with sample identification. 100 μl of the sample was added. Then in each tube was added 100 μl of SDS Solution. 2.5 ml of TBA/Buffer Reagent was forcefully down the side of each group and hence, the tubes were covered with glass marble and incubated at 95 °C for 60 min then removed from incubation and cooled to room temperature in an ice bath for 10 min. samples were centrifuged at 3000 rpm for 15 min. Finally removed supernatant from samples for analysis and read the absorbance at 532 nm.
Gene Expression
Total RNA was purified from the rat brain tissue using the RNAeasy® Lipid Tissue Mini Kit (Qiagen, Germany))Cat No. 74104 (. Purified total RNA from each sample was reverse transcribed by random hexamers of the High-capacity cDNA Reverse Transcription kit (Applied Biosystems, USA) for the preparation of complementary DNA (cDNA). The expression of GABAergic was estimated by quantitative RT-PCR (Light Cycler 480 II/96, Roche Applied Science, Switzerland) using iTaq™ Universal SYBR® Green Super mix kit, that was prepared according to the manufacturer’s protocol Gene Expression Assay (Assay ID Rn00691548_m1, Applied Biosystems, USA). Gene expression of glyceraldehyde-3-phosphate dehydro- genase (GAPDH) was used as a reference gene (assay ID Rn01775763g1, Applied Biosystems, USA). And specific primers were added to the reaction mix at a final concentration of 10 pM.
Microbial analysis:
Fecal collection and preparation for microbial analysis
In the present study, the fecal samples of the rats from all groups were collected in sterile tubes and kept at −20 °C until further use. The microbial analysis involved the culturing of microorganisms on different media and under different incubation conditions for their preliminary identification and enumeration indicating the alteration of the gut microbiota in response to the treatment being tested.
Fecal suspensions of each treated group correspondingly were prepared by dissolving 1:10 w/v in sterile phosphate-buffered saline (PBS, 0.1M) (Zhang et al., 2014).
All samples were homogenized using a sonicator for 5 s followed by centrifugation at 5000 rpm for 10 min at −4 °C. Ten-fold serial dilutions of the fecal suspensions were then performed. One milliliter of the supernatant from the original dilution (dilution 0) was added to 9 ml sterile PBS in a tube (dilution 1). The process was repeated until dilution 4 was created, and 0.1 ml of each of the prepared dilutions were loaded and spread on the surface of different culture media. The culture media used included nutrients agar were incubated aerobically at 37 °C for 18–24 h.
Bacterial enumeration and identification
Before incubation, the bacterial count from the different media was recorded as the colony count per plate. Data were compared between the rats' groups in the study. Preliminary bacterial identification was performed by morphological observation on the different media used. Further identification was made microscopically using the gram staining technique, where single colonies from the various culture media were selected, heated to form a smear,
subjected to a Gram staining procedure, and then observed under the microscope using an oil immersion lens.
Statistical analyses
The results of the present study were expressed as the means ± S.D. All statistical comparisons between the control group and the PA and probiotic-treated rats groups were performed using SPSS version 16.0. One-way analysis of variance (ANOVA) tests with Dunnett’s test for multiple comparisons was performed.
Table 1: Mean ±S.D. of all the measured variables in the brain homogenates of the six studied groups:
PPA+MIX
|
PPA+LAC
|
PPA+BIF
|
PPA+
|
PPA
|
Control
|
|
72.11 ± 18.05d**
|
58.74 ± 10.18d*
|
113.3 ± 16.67bc*
|
77.76 ± 14.39a**
|
58.41 ± 14.36a**
|
136.4 ± 16.95
|
Glutamine
|
2.033 ± 0.7631
|
1.214 ± 0.1813
|
2.773± 0.8548
|
1.931 ± 0.4517
|
1.061 ± 0.2552
|
3.883 ± 0.6326
|
Glutamate
|
278.7 ± 67.55b*
|
142.0 ± 29.95d*
|
347.8 ± 80.01bc*
|
152.3 ± 34.06a*
|
143.0 ± 24.02a**
|
283.5 ± 59.00
|
GABA
|
4.493 ± 2.594a*
|
1.324 ± 0.7396a*
|
6.479 ± 2.550bd*
|
3.096 ± 0.9080a**
|
1.752 ± 0.8360a**
|
9.912 ± 1.587
|
GABARA
|
47.09 ± 10.41a**
|
40.68 ± 6.111a**
|
33.30 ± 6.274ac**
|
51.24 ± 6.843a**
|
43.61 ± 5.181a**
|
10.17 ± 3.528
|
Lipid peroxides
|
90.67 ± 27.27bce*
|
27.39 ± 11.51
|
69.70 ± 45.15
|
41.16 ± 24.21
|
26.21 ± 15.94a*
|
81.62 ± 19.59
|
GSH
|
18.38 ± 5.644
|
6.135 ± 2.628
|
17.83 ± 6.741
|
9.010 ± 3.237
|
5.690± 3.775
|
18.55 ± 3.219
|
GST
|
(a) Control vs all groups, (b) PPA vs all groups, (c) PPA+ vs therapeutics group, (d) PPA+BIF vs
PPA+LAC and PPA+BIF vs PPA+MIX, (e) PPA+LAC vs PPA+MIX.
(*). The mean difference is significant at P˂ 0.001 level,
Table 2: Mean ±S.D. of all the measured variables Ratios of (I) GABA\GABARA, (II) GABA\Glutamate, (III) Glutamine\Glutamate) in the brain homogenates of the six studied groups:
PPA+MIX
|
PPA+LAC
|
PPA+BIF
|
PPA+
|
PPA
|
Control
|
|
39.86 ± 12.84b*
|
25.38 ± 11.18
|
30.90 ± 9.489
|
28.72 ± 13.32
|
16.18 ± 7.730
|
25.00 ± 9.945
|
Glutamine\glutamate
|
83.06 ± 44.28
|
61.63 ± 40.50
|
81.82 ± 28.73
|
64.26 ± 67.62
|
47.92 ± 19.39
|
83.27 ± 26.58
|
GABA\Glutamate
|
10.59 ± 5.425a*
|
11.21 ± 3.899a*
|
13.05 ± 8.337a*
|
12.64 ± 10.00a*
|
11.32 ± 5.397a*
|
25.32 ± 7.092
|
GABA\ GABARA
|
(a) Control vs all groups, (b) PPA vs all groups,
(*). The mean difference is significant at P˂ 0.001 level.
Table 3: Mean ± S.D of GABARA, GABARB, and GABARG selected subunits gene expression in brain homogenates of male western albino young rats, in all groups:
PPA+MIX
|
PPA+LAC
|
PPA+BIF
|
PPA+
|
PPA
|
Control
|
|
0.0371 ± 0.00073**
|
2.049 ± 0.0789*
|
2.493 ±0.291**
|
1.878 ± 0.086*
|
0.0716 ± 0.0089*
|
1 ± 0.409
|
GABARA1
|
0.0259 ± 0.0026**
|
0.310 ± 0.044*
|
1.574 ± 0.336*
|
0.924± 0.056401*
|
0.0116 ± 0.007**
|
1 ± 0.162
|
GABARA2
|
0.00059 ± 0.00015**
|
0.256± 0.0213**
|
1.132 ± 0.119**
|
0.648 ± 0.024**
|
0.00036± 5.18046E-05**
|
1 ± 0.027
|
GABARA3
|
0.019 ± 0.002**
|
0.142± 0.004**
|
0.683 ± 0.038**
|
0.564 ± 0.030**
|
0.0070 ± 0.0011**
|
1 ± 0.108
|
GABARA5
|
0.038± 0.00196**
|
0.677 ± 0.662
|
1.608 ± 0.269*
|
1.076 ± 0.135*
|
0.066 ± 0.0137*
|
1 ± 0.0195
|
GABARB2
|
6.81E-05 ± 8.92E-05*
|
0.561± 0.087*
|
2.099 ± 0.215**
|
1.089 ± 0.0064*
|
0.00025 ± 0.00011*
|
1 ± 0.158
|
GABARB3
|
0.01715± 0.0017*
|
0.2875 ± 0.0266*
|
1.181 ± 0.1609*
|
0.611 ± 0.028*
|
0.0410± 0.00322*
|
1± 0.199
|
GABARG2
|
(*). The mean difference is significant at P˂ 0.001 level,
(**) The mean difference is significant at P˂ 0.0001 level.
Table4: Estimation change of microorganisms in all groups. MCA, MacConkey agar; NA, Nutrient agar; MHA Mellur Henton agar; Blood agar. [(-): no growth, (+) Weak growth, (++): Medium growth, (+++): Strong growth.]:
Isolated Organisms
|
Media and incubation conditions
|
Control
|
PPA+
|
PPA + BIF
|
PPA + LAC
|
PPA + MIX
|
Enterobacteriaceae (Gram-negative rod, lactose fermenters)
|
MCA /Aerobic 37˚C/24hr
|
+
|
-
|
++
|
+++
|
++
|
Staphylococcus and/or Bacilli (Gram-positive cocci/ rod or Gram-negative rod)
|
N.A / Aerobic 37˚C/24hr
|
-
|
-
|
+++
|
+
|
++
|
Moraxella spp
Gram-negative
|
MHA / Aerobic 37˚C/24hr
|
++
|
+
|
++
|
++
|
++
|
Gram-pe/Gram-negative rod and positive cocci
|
Blood /Aerobic 37˚C/24hr
|
-
|
++
|
++
|
-
|
+
|
(-): No growth; (+): Weak growth; (++) Moderate growth; (+++) High growth