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Summer Research Fellowship Programme of India's Science Academies

Profile of anti-oxidant enzymes in cortex of Moderate Grade Hepatic Encephalopathic rat

Pritisha Mishra

Hindu College, University of Delhi 110007

Prof. S.K Trigun

Banaras Hindu University, Varanasi, Uttar Pradesh 221005

Abstract

Hepatic Encephalopathy (HE) is a set of neuropsychiatric complications arising out of liver dysfunction or damage causing motor and cognitive deficit due to persistent increase in the serum ammonia level (hyperammonemia: HA) and consequently in the brain. Since brain lacks the complete urea cycle enzymes, this persistent ammonia is metabolized by glutamate glutamine cycle in the brain resulting in increased extracellular glutamate accumulation in brainthereby causing over stimulation of the NMDA receptor and thereby induction of excitotoxicity in the brain. NMDAR overactivation at downstream level leads to oxidative stress and bioenergetic deficit which can be fatal. In this project, study of the antioxidant enzyme was conducted in cerebral cortex of the MoHE rats, developed by administration of 100 mg/kg bw of thioacetamide i.p. for 10 days. The initial studies undertaken for estimation of oxidative stress and energy deficit by measuring activity of lactate dehydrogenase (LDH) and adaptive changes in the redox-sensitive signaling pathways, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), suggested chronic oxidative stress development and cellular damage by ROS.

Keywords: hyperammonemia, NMDA, oxidative stress, energy deficit, antioxidant enzymes

Abbreviations

Abbreviations
HA Hyperammonemia 
HE Hepatic Encephalopathy 
bw  body weight
NMDA N-Methyl D-Aspartate 
NAFLD  Non- Alcoholic Fatty Liver Disease
 EAATExcitatory Amino Acid Transporters 
ROS Reactive Oxygen Species 
RNS Reactive Nitrogen Species 
CYP2E1 Cytochrome P450 2E1 
NBT Nitro Blue Tetrazolium 
PMS  Phenazine Methosulphate
TEMED N,N, N, N Tetramethylethylenediamine 
 APSAmmonium Per Sulphate 
TDW Triple Distilled Water 
 CCXControl Cortex 
 CLFChronic Liver Failure 
kDa Kilo Dalton 
GSH Reduced Glutathione 
GSSG Oxidised Glutathione 
SDS Sodium Dodecyl Sulphate 
PBST Phosphate Buffered Saline with Tween-20 
 CBBCoomassie Brilliant Blue 
HRP Horseradish Peroxidase 
SOD Superoxide Dismutase 
 LDHLactate Dehydrogenase 
GPx Glutathione Peroxidase 
 CLFChronic Liver Failure 

INTRODUCTION

Hepatic Encepthalopathy is a spectrum of neuropsychiatric complications arising out of liver dysfunction caused due to heavy alcoholism, viral infections(commonly, Hepatitis), NAFLD or cirrhosis. It manifests by a broad spectrum of neuropsychiatric disturbances such as: defects in cognitive, emotional, behavioural, psychomotor and locomotive functions ( R. Prakash, et al, 2010 ). Defective or inefficient Urea Cycle in hepatocytes results in HA which causes excessive toxic Ammonia to cross the blood-brain barrier. This disrupts the Glutamate-Glutamine cycle between neurons and astrocytes and reduced uptake of Glutamate by receptors on Astrocytes(EAAT1 and EAAT2) results in Glutamate accumulation in the cleft.

As a result, NMDA receptors on the post-synaptic neuron are overactivated and induce a cascade of downstream signaling. They open up excess of Ca2+ channels and high intracellular Calcium concentration has three effects on the neurons-

a. Bioenergetic Deficit

b. Oxidative Stress

c. Nitrosative stress

As a and b are the driving factors of pathogenesis of acute HE,

  • Profiling of Anti-oxidant enzymes’ levels
  • Assay of regulatory enzymes accountable to equip brain cells to utilize Lactate as alternate energy source during non-physiological conditions

are of paramount importance.

Low-grade-brain-edema-in-HE-Ammonia-induces-astrocyte-swelling-which-is-in-part.png
    Factors involved in the pathogenesis of HE

    Despite more than a century of research, the pathogenesis of HE is still not well understood. The most common suggestions include the role of neurotoxins(like Ammonia), impaired neurotransmission due to metabolic changes in liver failure, changes in brain energy metabolism, systemic inflammatory response and alterations of the blood brain barrier. 

    Statement of the Problem

    Data from both animal and human studies sustain that there is a synergistic effect between systemic oxidative stress, and ammonia in the pathogenesis of HE( Bosoi CR and Rose CF, 2013 )

    ROS accumulation, an important factor in HE pathogenesis overwhelms the antioxidant system of body and therefore, this project was aimed at evaluating the relationship between oxidative stress and pathogenesis of HE by monitoring and comparing profiles of SOD, CAT, GPx and LDH in brain tissue of Control rats and TAA- induced MoHE rats.

    Scope

    According to a recent WHO report,​ alcohol consumption has doubled in India and is incessantly increasing. Alcohol damages hepatocytes and leads to a spectrum of diseases such as Alcoholic Fatty Liver, Cirrhosis etc. This leads to a defective or inefficient Urea Cycle which brings about HA and ultimately HE conditions. Cirrhosis has a severe negative financial impact on the daily lives of their patients and their caregivers( Bajaj JS and Wade JB and Gibson DP and Heuman DM and Thacker LR and Sterling RK and Stravitz RT and Luketic V and Fuchs M and White MB and Bell DE and Gilles H and Morton K and Noble N and Puri P and Sanyal AJ, 2011).

    In India, Acute hepatic failure (AHF) mostly due to Hepatitis, almost always presents with encephalopathy and survival frequency of such patients is 33%. Most patients succumb to cerebral oedema or sepsis. 75% of deaths occur within the first 72 h of hospitalization, indicating that any intervention or novel management strategy for such patients must be encouraged. ( Acharya SK and Panda SK and Saxena A and Gupta SD, 2000)

    Thus, understanding the pathogenesis of HE in order to target appropriate factors for therapeutic intervention is a current requisite.

    LITERATURE REVIEW

    HE

    Liver is the largest gland in humans performing life-sustaining functions of storage, synthesis of metabolic requisites and most importantly, detoxification. When this functioning is impaired by virtue of cirrhosis(permanent scarring of liver), alcoholism or acute liver failure, it brings about a spectrum of neuropsychiatric abnormalities in the form of loss of cognitive, behavioural and psychomotor abilities blanketed under 'Hepatic Encephalopathy'.

    Stages of Hepatic Encephalopathy
    Stage  Symptoms
    I Mild confusion, irritability, agitation, sleep disturbance, decreased attention 
    II Lethargy, disorientation, inappropriate behaviour, drowsiness, Asterixis
     IIISlurred speech, marked confusion, aggression, somnolent but arousable, Asterixis 
    IV Coma 

    As hard scar tissue replaces soft, healthy tissue two physiological changes occur:

    -The scarred tissue cannot carry out detoxification as before.

    -The scarred tissue blocks blood flow through the liver causing high blood pressure in portal veins (portal hypertension).

    As a result, toxins like Ammonia, produced by the body when proteins undergo deamination or as a by-product of gut microbiome metabolism, build up. Ammonia is a neurotoxin responsible for HE development via direct effect on the metabolism and functions of the CNS and influencing the passage of various molecules across the blood-brain barrier, transport of branched chain amino acids, and aromatic amino acids (AA), whose inflow is augmented by exchange with glutamine produced in the course of ammonia detoxification. Disturbances of AA transport affect the brain catecholamine synthesis (serotonin and dopamine) and the production of “false neurotransmitters” (octopamine and fenylethylamine), resulting in impaired GABA-ergic, serotonergic, and glutamatergic neurotransmission ( Skowrońska M and Albrecht J, 2012 ).

    Thioacetamide (TAA)

    It is a water-soluble organosulfur compound has been used successfully to induce acute liver injury in rats, It is a potent centrilobular hepatotoxicant, which undergoes a two-step bioactivation mediated by microsomal CYP2E1 to thioacetamide sulphoxide (TASO), and further to a reactive metabolite thioacetamide-S, S-dioxide (TASO2) . It can release inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB), leading to centrilobular necrosis. (Chen TM and Subeq YM and Lee RP and Chiou TW and Hsu BG, 2008).

    download_1.png
    Structural formula of TAA
      F11.large.jpg
      Bioactivation of TAA in hepatocytes. (Adapted from: Potentiation of Thioacetamide Liver Injury in Diabetic Rats Is Due to Induced CYP2E1 , Wang et al., 2000) 
        TAA- A hepatotoxicant

        Glutamate-Glutamine Cycle and Astrocyte Swelling

        As brain lacks the five Urea cycle enzymes, majority of the metabolism occurs via Glutamine-Glutamate cycle which involves two key enzymes- Glutamine Synthetase(GS) and Glutaminase found in astrocytes and neurons respectively.

        Glutamate is a major excitatory neurotransmitter involved in primary perception and cognition,it’s pool being maintained by the Glutamate- Glutamine cycle in the synaptic cleft. Ammonia that reaches the brain is metabolized in astrocytes during conversion of Glutamate into Glutamine in a reaction catalysed by GS which is then released into the ECM to be then taken up by pre-synaptic neurons. In these neurons, Glutamate is again synthesized from Glutamine by enzyme Glutaminase. Glutamate synthesized is packaged into synaptic vesicles by Glutamate transporter( VGLUT) and released into the cleft. Glutamate binds to it’s receptors, bringing about Glutamergic Neurotransmission and is finally removed from the cleft by astrocytes.

        ketaminepcpcppt1336888009187-thumb400.jpg
          Glutamine- Glutamate Cycle

          NMDA Receptor

          NMDA receptors are Ca2+ sensitive ionotropic channels with the highest affinity for Glutamate than any other Glutamate receptor. It is one of the three ionotropic receptors- the other two being AMPA(alpha- amino 3-hydroxy-5- methylisoxazole 4-propionate) and KA(Kainic Acid) receptors.

          NMDA receptors are tetramers made up of 2 Glycine-binding NR1 subunits and two Glutamate binding NR2 or NR3 subunits. Most common are the receptors with two NR1 and two NR2 units. NR2 has further 4 subunits- NR2A, NR2B, NR2C and NR2D with NR2B primarily said to be involved in learning, memory and processing behavior.

          Continuous activation of these receptors leads to intracellular Calcium load which triggers a cascade of downstream effects that finally lead to Apoptosis or Necrosis. Effects include- Mitochondrial membrane depolarisation, ROS and RNS production and cellular toxicity.(Dong XX and Wang Y and Qin ZH, 2009 )

          fpsyt-04-00052-g001 (1).jpg
            NMDA Receptor structure. (Adapted from:  NMDA receptor activity in Neuropsychiatric disorders, Hadzimichalis and Lakhan et al., 2013)

            Oxidative Stress

            Oxidative stress is the imbalance between the oxidants produced and endogenous anti oxidant capacity of cells to prevent oxidative injury (Cadenas E, 1989). Brain consumes about 20% of the total oxygen of body due to highest oxygen metabolic rate and thus is a substantial source of ROS production.

            ROS and RNS are produced by normal cellular metabolism by action of enzymes such as NOS, NADPH oxidases etc with beneficial effects such as cytotoxicity against pathogens, intracellular messaging in cell differentiation, apoptotic and defensive pathways. Thus, low levels of ROS/RNS are constantly being produced in the body and are of great significance.

            Reactive-oxygen-and-nitrogen-species-a-Reactive-Oxygen-Species-ROS-Reactive-Nitrogen.png
              List of ROS and RNS 

              However, there is also present a robust anti-oxidant defence system in aerobic organisms whose intracellular levels are in a state of constant balance with ROS production for maintaining homeostasis. The different enzymatic and non-enzymatic anti-oxidant defences include Superoxide Dismutase(SOD), Catalase( CAT) , Glutathione Peroxidase ( GPx) reduced Glutathione (GSH) , Vitamin C( Ascorbic Acid) , Vitamin E ( Tocopherol) etc.

              LDH

              LDH catalyses the interconversion between Lactate and Pyruvate with the concomitant interconversion of NADH and NAD+. It catalyses NADH dependent conversion of Pyruvate(final product of Glycolysis) into Lactate in hypoxic conditions so as to regenerate NAD+ for Glycolysis. This is because Pyruvate cannot enter TCA and then ETC due to lack of oxygen. Due to lack of Oxidative Phosphorylation, the NADH:NAD+ ratio in hypoxic cells is high. As NAD+ is required in the preparatory phase of Glycolysis, Pyurvate is reduced to Lactate by LDH so as to ensure continued NAD+ production and indirectly, continued production of energy through Glycolysis.

              463d7e60f019c3acb2abf09aecfbe6507f8c01c3.png
                Pathway of LDH activity

                Thus, efficient production of Lactate is a common mechanism of bioenergetic adaptation in metabolically compromised brains(Ross et al., 2010). Mammals have 5 isoforms of LDH depending on the type of subunits present. LDH is tetrameric and the two subunits present are LDH-H protein and LDH-M protein encoded by LDHA and LDHB genes respectively. The random binomial combination of these 2 subunits result in 5 isoenzymes found in different tissues-

                LDH-1 (4H)- Heart, RBCs, Brain

                LDH-2(3H1M)- Reticuloendothelial system

                LDH-3(2H2M)- Lungs

                LDH-4(1H3M)- Kidney, Pancreas, Placenta

                LDH-5(4M)- Liver, Striated Muscle

                While the M subunit is predominant in anerobic tissues like striated(skeletal) muscles, the H subunit is predominant in aerobic tissues.

                Anti- Oxidant System

                PMC2715191_omcl0101_0015_fig001_1.png
                  Important anti-oxidants and their functioning

                  SOD

                  SOD( EC 1.15.1.1) is an enzyme that alternately catalyzes the dismutation (or partitioning) of the superoxide(O2-) radical into either ordinary molecular oxygen (O2) or hydrogen peroxide (H2O2) which is then further detoxified by specific enzymes. Superoxide is produced as a by-product of mitochondrial respiration and, if not regulated, causes cellular damage. Thus, SOD is an important antioxidant defense in nearly all living cells exposed to oxygen.

                  SOD has three isoforms-SOD-

                  1:Cytosolic Cu/Zn SOD, homodimer

                  2. SOD-2:Mitochondrial SOD( Mn- SOD) , homotetramer

                  3. SOD-3:Extracellular SOD( EC- SOD), heterotetramer

                  SOD destroys the superoxide radical by successive oxidation and reduction ( disproportionation) of the transition metal ion( Cu, Zn or Mn) at it’s active site by Ping pong mechanism. It has a molecular weight of 32.5 kDa.

                  While mice lacking SOD2 died several days post-birth due to massive Oxidative Stress ( Hu L and Ding M and Tang D and Gao E and Li C and Wang K and Qi B and Qiu J and Zhao H and Chang P and Fu F and Li Y, 2019 ) and those lacking SOD1 developed pathological conditions like hepatocellular carcinoma, mice lacking SOD3 did not show any defect and had a normal life span.

                  9ff1.jpeg
                    Isoforms of SOD

                    Catalase (EC 1.11.1.6)

                    It is a tetrameric enzyme consisting of 4 identical tetrahedrally arranged sub-units containing a single Ferritoporphyrin group each. It has a molecular mass of 240 kDa. It is found primarily in peroxisomes(a membrane- enclosed cell organelle) and detoxifies H2o2 by catalyzing a reaction between two Hydrogen peroxide molecules, resulting in the formation of water and O2.

                    It can also catalyse interaction of H2O2 with H-donating compounds(alcohol like ethanol) resulting in 1 H2O molecule and oxidized form of the compound.

                    Glutathione peroxidase (EC 1.11.1.19)

                    It is the general name of an enzyme family with peroxidase activity whose main biochemical function is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water by oxidizing GSH to GSSG.It has a molecular weight of 80 kDa and has a Selenocysteine residue in each of the four identical subunits. There are 8 GPx isoenzymes in mammals with the levels of each isoform varying with location.

                    MATERIALS AND METHODS

                    Experimental Design

                    Adult male rats weighing between 160-170g were used in this experiment . They were housed in separate cages and fed food pellets and water ad libitum. Two different groups of rats were taken, each group having a minimum of 6 rats- Control and TAA-exposed.

                    For the experiment, the control rats were injected intra-peritoneally with 0.9% 1.5 M NaCl for a period of 10 days while simultaneously, the TAA-exposed rats were injected intra-peritoneally with 100 mg/kg body weight TAA(dissolved in 1.5 M NaCl)

                    Sample Collection

                    At the end of experimental period, the rats were sacrificed and dissected by Transcardial Perfusion. The brains were collected in chilled 4% PFA and stored in – 80°C for further use.

                    · For study of Histology of the brain tissue of 2 groups, the brains after being collected in chilled 4% PFA were given 2 changes of PFA and stored overnight at 4ºC. Tissue was then washed with 1x PBS and stored in increasing grades of sucrose at 4ºC.After this, the brain is embedded in OCT medium in Aluminium foil vials using liquid nitrogen and Isopentane and stored at -80ºC.

                    Homogenate Preparation and Protein Estimation

                    15% homogenate(150 mg in 1 ml buffer) of 2 different groups- Control and TAA- exposed rats was prepared by differential centrifugation.

                    Materials

                    1. Extraction Buffer- comprising 50 mM pH- 7.4 Tris Cl, 100 mM KCl, 100 mM MgCl2, 1 mM EGTA, 50 mM HEPES, 100 mM Sucrose and TDW to make up the volume.

                    2. PR(Protein Reagent)= 2% Na2CO3- 0.1 N NaOH + 1% CuSO4 + 1% Na+ - K+ Tartarate in the ratio 100:1:1

                    3. Folin’s Reagent= Folin-Ciocalteu reagent and TDW in 1:1 ratio.

                    Protocol

                    Method used for protein estimation is Lowry’s method, a modified version of Biuret reaction. In this, Cu2+ ( from CuSO4 of PR) forms a coordinate tetravalent compound with peptide bonds under alkaline conditions resulting in it’s reduction to Cu+ (similar to that in Biuret reaction). Cu+ then reacts with Folin- Ciocalteu( Phosphomolybdate- Phosphotungstate) reagent to form a bluish coloured compound, hetero-polymolybdenum blue which is assessed spectrophotometrically at 660 OD. Intensity of colour depends on amount of Aromatic AAs present- phenolic(-OH) group of such AAs reduces the phosphomolybdotungstate to the bluish complex and this reaction is catalysed by Cu+

                    a. 150 mg tissue was weighed( 50 mg from each of the 3 brain samples) and collected in 1ml extraction buffer.

                    b. The samples were homogenized using Potter- Elvejham homogenizer ( 10x * 3 strokes) and centrifuged in a pre-cooled centrifuge for 10 mins at 4 °C and 850 rpm.

                    c. Pellet formed was discarded and supernatant was collected.

                    d. Supernatant was centrifuged at 1000 rpm for 10 mins and degrees to obtain the nuclear pellet.

                    e. Supernatant collected was again centrifuged at 10000 rpm for 30 mins to obtain mitochondrial pellet. Supernatant pipette out is the cytosolic extract.

                    f. To obtain mitochondrial extract, the pellet was dissolved in extra extraction buffer and centrifuged at 10000 rpm for 10 mins at 4°C.

                    g. The extract tubes were placed in -80°C until further use.

                    h. The samples were taken in triplicates along with a blank. 4µl sample was diluted 25 times with TDW( 4µl + 96 µl )

                    i. To sample mixture, 1 ml Protein Reagent(PR) was added and incubated in dark for 10 mins.

                    j. To that, 100 µl Folin’s Reagent was added and incubated in the dark for 30-40 minutes.

                    k. The sample mixture was read spectrophotometrically at 660 OD and amount of protein( µg) was calculated using the formula-

                    Observed OD* Standard OD* Dilution Factor

                    In-gel Activity Assays

                    While SDS–PAGE is the most frequently used gel system for separating proteins on basis of charge to mass ratio,it can’t be used to detect a protein(enzyme) on basis of it’s biological activity. In this case, native PAGE is used which provides non-denaturing and constant-pH conditions. Polyacrylamide gels are again used sans SDS and thus proteins are not denatured prior to loading. Since all the proteins being analysed carry their native charge at the pH of the gel (7.4), proteins separate according to their different electrophoretic mobilities and the sieving effects of the gel, charges and sizes of proteins in a given protein mixture, good resolution is achieved.

                    2 types of buffer systems can be used:

                    a. Continuous- Same buffer is used in gel and electrophoresis tank. No separate gels used which provides a uniform separation matrix. Used mainly for Nucleic Acid analysis.

                    b. Discontinuous- Different buffers used in gel and electrophoresis tank. 2 kinds of gels used-

                    Stacking Gel: The upper gel with lower acrylamide concentration poured atop resolving gel where combs are inserted to obtain wells. It has a very large pore size which allows the proteins to move freely without any prominent frictional drag and stack into a sharp band under the effect of the electric field.

                    Resolving Gel: The smaller pore size by virtue of higher Acrylamide percentage introduces a sieving effect which separate proteins on basis of molecular size. Higher the kDa of protein to be separated, lower is the percentage of gel(8% or 10%), i.e larger the pore size. Thus, the larger proteins can pass unhindered.

                    IMG_20190620_143937709_2.jpg
                       A Native- PAGE being run at 4°C

                      Materials and methods

                      1.  30% acrylamide: 29.22g acrylamide+ 0.78g N, N – methylene bis-acrylamide

                      2. 10% APS: provides the free radical necessary for the catalysis of the Polymerization of Acrylamide and Bis-acrylamide.

                      3. TEMED:Catalyses reaction of ammonium persulfate to form free radicals, TEMED accelerates the polymerization of acrylamide and bis-acrylamide.

                      4.Tris- glycine electrophoresis buffer from 5x stock: 500 mL

                      5.2x Native Dye: 1 ml Glycerol + 1 ml Tris Cl(pH-7.4) +1 pinch Bromophenol Blue. It forms a dye front ahead of the sample and gives us an idea of the distance migrated by it on the gel.

                      6. TDW

                      A. LDH Assay:

                      • Materials:

                      1. LDH Staining Solution(20 ml) :

                      a. Lithium Lactate(0.1 M)- 192 mg

                      b. NAD+ (1 mg/ml)- 20 mg

                      c. NBT(0.25 mg/ml)- 5 mg

                      d. NaCl (0.1 M)- 1 ml

                      e. MgCl2( 0.5 mM)- 10 µl

                      f. 1M Tris-Cl(pH- 7.4) - 2.5 ml

                      g. PMS(0.25 mg/ml)- 0.5 mg

                      • Protocol:

                      60 μg protein sample was loaded to a 8% native polyacrylamide gel, diluted with equal volume of 2x Native Dye( volume as estimated by Lowry’s) in the sequence- CCX followed by CLF for 6 wells. Gel, after being pre-run( to remove residual or unpolymerised components of gel) ,was first run at 50 V for 30 mins and then at 100 V till dye is completely over-run. The gel was then taken out and stained in the staining solution.

                      Upon appearance of bands, the staining solution was discarded and gel washed with TDW before scanning.The bands were quantitated by gel densitometry using AlphaImager 2200 gel documentation software.

                      • Principle:

                      LDH oxidises the substrate( Lithium Lactate) and forms Pyruvate utilising the NAD+ provided in the mixture. NADH so formed forms a complex with NBT, in presence of PMS, to form a purplish compound called Formazan. MgCl2 provides Magnesium ions which act as a cofactor of LDH. Thus, as a result, purplish bands are formed where LDH is present on the gel when gel is placed in staining solution. Where LDH is absent, no bands are seen. The intensity of purple bands is directly proportional to the amount of LDH present. As a 8% gel is used, the different isozymes of LDH are also distinctly separated.

                      LDH princi_1.jpg
                        Mechanism of LDH assay

                        B. Catalase Assay

                        • Material:

                        a. 0.03% H202 solution- 3 µl H202 diluted upto 20 ml with TDW.

                        b. Freshly prepared 50:50 solutions of 2% Potassium Ferricyanide(0.2 g in 10 ml TDW) and 2% Ferric Chloride-(0.2g in 10 ml TDW)

                        After gel is removed from solution a, both components of b are added simultaneously onto gel.

                        • Protocol:

                        60 µg sample was loaded onto 8% Native Polyacrylamide Gel in a total of 6 wells- in an alternate sequence of CCX and CLF. The gel, after pre-run, was run for 4-5 hours, first 30 mins at 50 V and henceforth at 100V. After that, gel was overrun for 30 minutes. The gel was then incubated in solution a for 25-30 minutes and then in solution b followed by manual shaking for 30-60 seconds and scanned immediately.

                        • Principle:

                        Catalase, wherever present on gel catalyses disproportionation of H2O2 into H20 and oxygen. Thus, pale yellow bands appear against the dark Prussian background of gel where due to absence of enzyme, Potassium ferricyanide gets reduced to ferrocyanide in presence of substrate(H2O2) which then reacts with Ferric chloride to form insoluble, stable Prussian blue complex.

                        C. SOD Assay

                        • Material:

                        Staining solution – 0.25 mM NBT , 28 microM Riboflavin and 28 mM TEMED with TDW to make up the required volume.

                        • Protocol:

                        60 µg sample was loaded onto 10% Native Polyacrylamide Gel in a total of 6 wells- in an alternate sequence of CCX and CLF. The gel was first pre-run at 50 V for 30 minutes and then run for 3-4 hours, first 30 mins at 50 V and henceforth at 100V. It was then stained in a staining solution for 30 mins while put under an illuminated lamp. Upon appearance of white bands against a dark purple background, the solution was discarded and the gel was scanned. The bands were quantitated by gel densitometry using AlphaImager 2200 gel documentation software.

                        • Principle:

                        This assay is an example of negative gel staining. In this, NBT, a competitor for superoxide radical is used as a color indicator. Exposure to light causes Riboflavin to generate superoxide radicals in presence of oxygen and TEMED. NBT and SOD compete for the same radicals and thus, where SOD is present in the gel, the gel remains transparent whereas in the remaining portion, the gel turns dark purple due to reduced NBT.

                        D. GPx Assay

                        • Material:

                        a. 50 mM Tris Cl buffer of pH 7.9 – 1 ml

                        b. 3 mM GSH- 20 mg

                        c. 0.004% H2O2 – 1 μl

                        (Volume made upto 20 ml with TDW)

                        a. 1.2 mM NBT- 19.61 mg

                        b. 1.6 mM PMS- 9.8 mg

                        (Volume made upto 20 ml with TDW)

                        • Protocol:

                        60 µg sample was loaded onto a 10% Native Polyacrylamide gel in a total of 6 wells- in an alternate sequence of CLF and CCX. The gel was first pre-run for 30 minutes at 50 V and then run at 50 V for 30 minutes followed by 100V. When dye reaches near Agarose sealing, gel is removed and incubated in solution a for 25-30 minutes followed by addition of solution b. Gel is scanned when bands begin to appear.

                        • Principle:

                        GPx catalyses disproportionation of H2O2 to H2O and O2 by oxidizing GSH. In absence of GSH, H202 reduces NBT by first reducing PMS to form blue formazan complex. Thus, achromatic bands reflect GPx activity against blue background due to formazan.

                        Lactate Assay

                        • Materials: This assay is done via Lactate liquid assay kit containing 2 solutions- a reagent and a Standard(30.9 mg/dl). The reagent contains-

                        - pH 7.6 buffer

                        - 1 mmol/L TOPS

                        - 0.25 mmol/L 4-Aminoantipyrine

                        - Lactate Oxidase(> 100 units/L)

                        - Peroxidase(> 100 units/L)

                        - Stabilisers, activators, preservatives and surfactants

                        • Protocol:

                        In a 96-well microtitre plate-

                        Assay composition
                         Components Blank(µl)Standard (µl)Sample(µl) 
                        TDW 2.5 
                         Standard solution2.5 
                        Homogenised sample 2.5 
                        Enzyme reagent 250 250 250 

                        Mix well and take reading at A= 550 nm using multi-plate reader.

                        • Principle:

                        Lactate Oxidase promotes oxidation of Lactic Acid to Pyruvate( Anaerobic Glycolysis) and H202. Then, peroxidase catalyses reaction of H2O2 with a H-donor(TOPS) in presence of 4-Aminophenazone to form a pinkish red product(Chinominin dye)which is measured spectrophotometrically at 550 nm.

                        Lactate + O- Lactate Oxidase → Pyruvate + H2O2

                        H2O2 + TOPS + 4 Aminoantipyrine→ Chinonimin + H2O

                        SDS-Page

                        • Materials:

                        1. Electrophoresis(running) buffer(pH- 8.3)- 500 ml-

                        Running buffer preparation
                         1. Add 100 ml Tris Glycine
                         2. Make up volume upto 495 ml with TDW
                         3. Add 5ml 10% SDS

                        2. Gel preparation-

                        12% Resolving Gel Preparation(5 ml)
                         ComponentConcentration(ml) 
                         TDW1.6 
                         30% Acrylamide mix 1.7
                         1.5M Tris( pH 8.8) 1.3
                         10% SDS 0.05
                         10% APS 0.05
                         TEMED 0.002
                        5% Stacking Gel Preparation( 3 ml)
                         ComponentConcentration(ml) 
                         TDW2.1 
                         30% Acrylamide mix 0.5
                         1.0 M Tris( pH 6.8)0.38 
                         10% SDS0.03 
                         10% APS 0.03
                         TEMED 0.003

                        3. 2x SDS dye- 1 ml;

                        400 µl SDS + 300 μl Gycerol + 200 microl TDW + pinch of Bromophenol Blue+ 14.2 µl β-Mercaptoethanol + 100 µl pH 6.8 Tris Cl buffer

                        4. Staining solution- Methanol: TDW: GAA (5:4:1 ) + 2.5 g CBB CBB staining

                        5. Destaining solution- Methanol:TDW:GAA(5:4:1)

                        6. Ponceau S reagent- 0.5 g Ponceau S dissolved in 1 ml Acetic Acid and volume made up with TDW till 100 ml.

                        7. Transfer buffer- 1 L; Tris- 3.03 g + Glycine- 14.4 g + Methanol- 100 ml + 10% SDS- 1 ml

                        8. PBST- 0.5% Tween-20 in 1x PBS

                        9. 4% Milk(Blocking) solution- 4g milk powder in 100 ml 1x PBS

                        10. Antibody solution- To 3ml 5% milk solution, 3µl 1 Ab added.( 1:1000 dilution) . Similarly, to 3 ml 5% milk solution, 3 µl 2 Ab added( 1:1000 dilution)

                        11.Developer

                        12.Fixer

                        13. ECL- Substrate: H2O2and Luminol in 1:1 ratio ; 2 ml

                        14. AgX coated- Xray film

                        • Principle:

                        SDS-PAGE is a discontinuous and denaturing PAGE used primarily to separate protein molecules on basis of their molecular size only. The proteins are assigned an uniform negative charge( by virtue of SDS anionic detergent) and are denatured into their primary structure by virtue of SDS and beta- mercaptoethanol. Laemelli buffer system is mostly used where relative migration of glycinate, chloride and negatively charged proteins resulting in an ‘Ion Front’ plays an important role.

                        IMG_20190603_135916337.jpg
                          SDS-PAGE being run at room temperature

                          • Protocol:

                          1. CBB Staining(Validation for Western Blot)

                          This is done to profile all the proteins present in sample after electrophoresis. The dye binds to proteins through ionic interactions between dye sulfonic acid groups and positive protein amine groups as well as through Van der Waals attractions.

                          1. A 10% SDS- PAGE gel was cast and allowed to polymerise. Gel was set onto electrophoretic tank filled with running buffer.

                          2. Samples( diluted with 2x SDS dye in 1:1 ratio) were boiled for 5 minutes followed by cold shock( at -20°C) immediately. (Protein denaturation step)

                          3. After gel is run at 50V for 30 minutes and then at 100V for 2-2.5 hours, gel was transferred to staining solution and put on rocker overnight.

                          4. Gel was destained with destaining solution 2-3 times for 2-3 hours until bands appeared.

                          2. Western Blotting and Immunodetection

                          This is a technique to separate out a specific protein from a sample mixture and visualizing it on a X-ray film via Antigen-Antibody interaction.

                          1. SDS-PAGE:

                          Samples are diluted with 2x SDS dye in the ratio 1:1 and loaded onto a 12% gel after heat-shock treatment. The gel is set in the electrophoretic tank filled with Running buffer and is run first at 50V for 30 mins and later at 100V for 2.5-3 hours.

                          2. Blotting: Wet transfer by electrical blotting

                          • For this, gel was first equilibrated in transfer buffer for 30 mins and Nitrocellulose membrane dipped with TDW.
                          • The negatively charged gel(placed towards black colorcoded anode of tank) and neutral membrane(towards red colorcoded cathode) were sandwiched between pre-wetted scrubbers, layers of Whatman paper and foam within the cassette of transfer apparatus containing transfer buffer.
                          • Transfer was set at constant current(70 mA) overnight (12-18 hours) at 4 °C

                          3. Transfer efficiency check by Ponceau S:

                          To check and confirm presence of protein bands, the membrane was dipped in Ponceau S reagent. Upon appearance of bands, membrane was washed with TDW.

                          4. Blocking by 4% Milk solution

                          To prevent non-specific binding of Antibodies to the remaining(free) membrane surface in subsequent steps, the membrane is incubated in 4% milk solution for 1-2 hours on a rocker. This is important as Nitrocoellulose membrane has a high affinity to proteins.

                          5. Primary Antibody treatment:

                          Specific 1° Ab(Monoclonal rabbit anti-Mn SOD and Anti- β Actin) diluted with 4% milk solution in 1:1000 ratio(3 μl to 3 ml milk solution) was added to the cellophane packet containing membrane . It was first placed on rocker for 1-2 hours and then kept overnight at 4°C

                          6. Secondary Antibody treatment:

                          After washing with PBST, the membrane was treated with HRP- conjugated 2°Antibody( Anti-rabbit goat) diluted with 4% milk solution in 1:1000 ratio( 3 microl to 3 ml milk solution) and kept on rocker for 2 hours. The 2° Ab is specific for 1° Ab and is linked to an enzyme( For example, HRP or Alkaline Phosphatase)

                          7. Immunodetection in dark room:

                          • The membrane was subject to a PBST-wash for 15-30 seconds and then processed for immunodetection using ECL detection kit. Membrane was exposed to a specific substrate(H2O2) diluted with Luminol in 1:1 ratio. Substrate and Luminol placed in dark bottles. Both were mixed under red light after a brief exposure of 30 s for charging Luminol.
                          • Membrane placed within cassette enclosed in a cellophane polybag and atop it, X-ray film(cut to desired length) placed.
                          • After a very brief exposure time, X-ray film dipped in Developer solution very briefly and exposed to red light
                          • X-ray film dipped in water and then dropped into Fixer solution.

                          8. Data Analysis:

                          The X-ray film was scanned and bands were quantitated by densitometry using Alpha imager software.

                          1.5   Cryosectioning and Nissl Staining (Wu et al., 2003):

                          Materials

                          a. 0.5% Cresyl Violet solution- 250 mg Cresyl violet was taken in a falcon and volume made upto 50 ml with autoclaved DW and a few drops of Glacial acetic acid was added.

                          b. OCT medium- Contains polyethylene glycol and polyvinyl alcohol

                          c. Liquid nitrogen and Isopropanol

                          d. Gelatin

                          Protocol

                          OCT-embedded tissue was fitted on the chuck by placing them on the Cryobar of a cryomicrotome(maintained at -20º) and the chuck was then fixed on the rotor. Coronal sections of 25 microns were cut on 3% Gelatin-coated slides and stored at -80º. For Nissl staining, sections were first thawed and air-dried. Slides were dipped in 1:1 mixture of chloroform and absolute ethanol overnight (de-fattening step) and then the sections were hydrated by downgradation:

                          1.     100% ethanol ( 5mins)  
                          2.     95% ethanol ( 5 mins)
                          3.     70% ethanol ( 5 mins)
                          4.     50% ethanol( 3 mins)
                          5.     30% ethanol (2 mins)
                          6. TDW( 2 mins)

                           

                          The sections are then stained with 0.5% Cresyl Violet solution for 45 minutes at 37-50 degrees. This was followed by differentiating the sections very briefly in 95% ethanol+ 5 drops GAA and quick dehydration in absolute ethanol. 2 changes of Xylene, 5 minutes each was given to the slides followed by mounting in DPX and leaving them overnight to dry. Slides were observed the next day under the microscope and imaging was done for analysis.

                          Principle

                          Nissl stain allows differential staining of brain tissue and thus study of morphology of structures in different conditions. The Nissl substance (RER), abundant in neurons’ cytons, is darkly-stained by cresyl violet, a basic aniline dye. Initially, tissue sections are "defatted" by placing in ethanol and chloroform overnight and then, rehydrated by passing back through decreasing concentrations of ethanol and lastly into water. The ethanol solutions act to differentiate the stain, causing myelin and other components to lose color whereas cytons retain the color.

                          RESULTS AND DISCUSSION

                          The results are being summarized as below

                          · Lactate levels were found to be elevated in the cortex of TAA treated rats.

                          · LDH: The level was found to be elevated in TAA-treated cortex than control cortexof rats.

                          · SOD: Level of both Cytosolic as well as mitochondrial SOD wasfound to be elevated in TAA-treated cortex than control cortex of rats.

                          · Catalase: The level was found to be elevated in TAA-treated cortex than control cortex of rats.

                          · GPx: The level was found to be elevated in TAA-treated cortex than control cortex of rats.

                          Lactate Assay

                          newgraphs.png
                            Lactate levels in Control vs TAA-treated groups. Results are expressed in terms of values representing Mean±SD where n=3 and probability(p) * < 0.05

                            In-gel LDH Activity Assay

                            LDH 101.png
                            • 1
                            • 2
                            LDH_3.png
                            Densitometric analysis of LDH levels in Contol vs CLF rat cortex.
                              Fig 13 Effect of TAA Chronic liver failure on brain LDH levels. Results are expressed in terms of values representing Mean±SD where n=3 and p***<0.001.

                              In-gel SOD Activity Assay

                              SOD101.png
                              Scanned representative image of 10% non-denaturing PAGE for SOD activity out of 3 PAGE repeats.
                              • 1
                              • 2
                              sod_3.png
                              Densitometric analysis of LDH levels in Control vs CLF rat cortex
                                Effect of Chronic liver failure on SOD levels in cerebral cortex. Results are expressed in terms of values representing Mean± SD where n=3 and p***<0.001

                                In- Gel Catalase Activity Assay

                                CATALASE101.png
                                Scanned representative image of 8% non-denaturing PAGE for Catalase activity out of 3 PAGE repeats.
                                • 1
                                • 2
                                Catalase_3.png
                                Densitometric analysis of LDH levels in Control vs CLF rat cortex.
                                  Effect of Chronic liver failure on Catalase activity in cerebral cortex of rat. Results are expressed in terms of values representing Mean± SD where n=3 and p***<0.001

                                  In- Gel GPx Activity Assay

                                  gpx101.jpg
                                  Scanned representative image of 10% non-denaturing PAGE for GPx activity out of 3 PAGE repeats.
                                  • 1
                                  • 2
                                  • 3
                                  • 4
                                  • 5
                                  GPx_1.png
                                  Densitometric analysis of LDH levels in Control vs CLF rat cortex. 
                                    Effect of chronic liver failure on GPx activity in cortex. Results are expressed in terms of values representing Mean± SD where n=3 and p***<0.001

                                    Western Blot of Mn-SOD

                                    CBB staining.png
                                    CBB Staining for protein profiling
                                      western.png
                                      Scanned image of Western blot for Mn-SOD vs that of β-Actin 
                                        Western Blot Analysis of Mitochondrial SOD in Control and CLF rat brains.

                                        Histology

                                        image.tif
                                        1
                                          image0022.tif
                                          2
                                            image0008.tif
                                            3
                                              image0006.tif
                                              4
                                                image0007.tif
                                                5
                                                  image0011_1.tif
                                                  6
                                                    Morphological changes in cerebral cortex tissue of Control(1-3) vs CLF rats(4-6) A. 10x view of Hippocampi of Control(1) vs CLF(4) groups. B. 40x view of CA1 region of Hippocampi of Control(3) vs CLF(5) group. C. 40x view of CA3 region of Hippocampi of Control(4) vs CLF (6) group.

                                                    It has been quite evident that oxidative stress prevails under cirrhotic or chronic liver damage conditions. To overcome the deleterious effects of ROS and RNS overproduction, several anti- oxidant enzymes get activated and their activity increases.

                                                    TAA induction in rats causes chronic liver damage which leads to HA. Ammonia enters brain by crossing the blood-brain barrier and brings about HE conditions.This leads to oxidative stress as is confirmed by elevated levels of major anti-oxidant enzymes- SOD, GPx and Catalase.This is further supported by the Western blot of Mn-SOD,localised in mitochondria and involved in fighting oxidative stress which shows elevated Mn-SOD expression.

                                                    SOD is the first committed enzyme of the anti-oxidant system and helps overcome ROS production by catalyzing conversion of superoxide ion into hydrogen peroxide which is then reduced into harmless metabolites by two major enzymes- Catalase and GPx(Fig 4). Thus, increased levels of SOD subsequently leads to elevated levels of Catalase and GPx in cortex tissue of CLF rats which corresponds to the fact that CLF does, indeed, induce oxidative stress in the brain which might lead to development of MoHE.

                                                    Elevated levels of Mn-SOD might also lead to oxidative burst of Mitochondria.

                                                    Production_of_mitochondrial_ROS,_mitochondrial_ROS.png
                                                      Routes for escape of ROS during Oxidative Phosphorylation in inner mitochondrial membrane.

                                                       

                                                      Major source of energy in brain, like in other tissues, is through Electron Transport Chain(ETC) occurring in the inner mitochondrial membrane. However, the pathway allows escape of superoxide radicals at certain junctions when produced in abundance which leads to over expression of Mn-SOD so as to counter balance ROS production(Fig 17b). As a result, aerobic respiration is hindered which subsequently leads to energy deficit and a tendency to shift to anerobic glycolysis.

                                                       As the cells cannot continue aerobic respiration due to dysfunctional ETC (and hence reduced pool of NAD+), the neurons shift to anaerobic glycolysis which is supported by two observations

                                                       a. Elevated LDH and Lactate(substrate for Anaerobic glycolysis) levels which leads to high NADH:NAD+ ratio.

                                                      b. Of all isoforms of LDH, M4, expressed mainly in tissues subject to frequent anerobic glycolysis, shows the most distinct change between the two experimental groups.

                                                      Thus, TAA induced CLF leads to development of oxidative stress and subsequent bioenergetic deficit in brain cortex which has major implications in the pathogenesis of MoHE.

                                                      CONCLUSION

                                                       The objective of this study was to determine the effect of liver damage on brain metabolism and was done by mainly focusing on Oxidative stress and Bioenergetic deficit profiling. As suggested by the results,levels of anti-oxidant enzymes in brain cortex are enhanced in case of liver damage which may lead to generation of several neurological and pathological conditions blanketed under "Hepatic Encephalopathy".

                                                      Other techniques learnt-

                                                      • Cryosectioning
                                                      • Paraffin block preparation
                                                      • Primer designing
                                                      • Vibratome sectioning
                                                      • Cell culture- Streaking, sub-culturing and seeding
                                                      • RNA Isolation and RT-qPCR
                                                      • Immunofluoroscence

                                                       

                                                       

                                                      Source

                                                      • Fig 1: https://www.researchgate.net/figure/Low-grade-brain-edema-in-HE-Ammonia-induces-astrocyte-swelling-which-is-in-part_fig1_221881872
                                                      • Fig 2 a: Wikipedia.org
                                                      • Fig 2 b: http://jpet.aspetjournals.org/content/294/2/473
                                                      • Fig 4: https://www.frontiersin.org/articles/10.3389/fpsyt.2013.00052/full
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