ATP, adenosine triphosphate, is the energy storage molecule used by cells in the
body. Within the nervous system its main role is to provide energy for active transport of
sodium and potassium ions to produce an electrical charge difference between the inside
and outside of the neurons. This 'action potential' is the means of nerve transmission. ATP
is a neurotransmitter in the central and peripheral nervous systems and is also involved in
peripheral inflammation and transmission of the sensation of pain. ATP plays three major
roles: it acts as an inhibitory transmitter from the enteric motor neurons to the smooth
muscle: it's also released as an excitatory neurotransmitter between enteric interneuron’s
and from the interneuron to the motor neurons: Finally, ATP may act as a sensory
mediator, from epithelial sources to the intrinsic sensory nerve terminals. ATP participates
in the transduction of sensory stimuli from the gut lumen and in the subsequent initiation
and propagation of enteric reflexes.
In a similar way, Adenosine diphosphate, abbreviated as ADP, is an important
organic compound in metabolism and is essential to the flow of energy in living cells. The
two phosphates in ADP can be correlated with ATP and AMP. ATP consists of three
phosphate groups attached in series to the 5’ carbon location, whereas ADP contains two
phosphate groups attached to the 5’ position, and AMP contains only one phosphate group
attached at the 5’ position. Adenosine monophosphate (AMP), also known as 5'-adenylic
acid, is a nucleotide that is used as a monomer in DNA and RNA.
Regulation of the ionic environment of a cell is important for cell function, in
particular during embryogenesis and in the nervous system. Energy transfer used by all
living things is a result of dephosphorylation of ATP by enzymes known as ATPases.
These are a class of enzymes that catalyze the decomposition of ATP into ADP and a free
phosphate ion. This dephosphorylation reaction releases energy, which the enzyme (in
most cases) harnesses to drive other chemical reactions that would not otherwise occur.
This process is widely used in all known forms of life.
There are different types of ATPases as given below which can differ in function
structure and in the type of ions they transport.
89 i) F-ATPases (F1FO-ATPases) in mitochondria, chloroplasts and bacterial plasma
membranes are the prime producers of ATP, using the proton gradient generated by
oxidative phosphorylation (mitochondria) or photosynthesis (chloroplasts).
ii) V-ATPases (V1VO-ATPases) are primarily found in eukaryotic vacuoles, catalysing
ATP hydrolysis to transport solutes and lower pH in organelles like proton pump of
lysosome.
iii) A-ATPases (A1AO-ATPases) are found in Achaea and function like F-ATPases
iV) P-ATPases (E1E2-ATPases) are found in bacteria, fungi and in eukaryotic plasma
membranes and organelles, and function to transport a variety of different ions across
membranes.
V) E-ATPases are cell-surface enzymes that hydrolyse a range of NTPs, including
extracellular ATP.ATPases were also classified based on the requirement of specific
cations such as Na+/K+ -ATPase, Mg2+ -ATPase and Ca2+ -ATPase.
Na+/K+ -ATPase (sodium, potassium-adenosine 5’-triphosphate):
Na+/K+-ATPase is an antiporter enzyme (EC 3.6.3.9) located in the plasma
membrane of all animal cells. The Na+-K+ -ATPase enzyme pumps sodium out of cells,
while pumping potassium into cells. Active transport is responsible for cells' containing
relatively high concentrations of potassium ions but low concentrations of sodium ions.
The mechanism responsible for this is the sodium-potassium pump, which moves these
two ions in opposite directions across the plasma membrane which was known by
radioactively labeled ions across the plasma membrane of certain cells. It was found that
the concentrations of sodium and potassium ions on the two sides of the membrane are
interdependent, suggesting that the same carrier transports both ions. It is now known that
the carrier is an ATPase and that it pumps three sodium ions out of the cell for every two
potassium ions pumped in. The levels of Na+ and K+ ions in the cell are determined, in
part, by the action of membrane-bound Na+, K+-ATPase [ATP phosphohydrolase (Na+/K+90 transporting); EC 3.6.1.37] or the sodium pump. This enzyme is composed of two
subunits, a 95,000-100,000 Mr α subunit and a 45,000-60,000 Mr β subunit. The α or
catalytic subunit contains the ion channel and the ion-, ATP-, and cardiac glycoside (i.e.,
ouabain)-binding sites characteristic of the enzyme.
The Na+-K+ ATPase helps maintain resting potential, avail transport, and regulate
cellular volume [Hall et al., 2006]. It also functions as signal transducer/integrator to
regulate MAPK pathway, ROS, as well as intracellular calcium. In most animal cells, the
Na+-K+ ATPase is responsible for about 1/5 of the cell's energy expenditure. For neurons,
the Na+-K+ ATPase can be responsible for up to 2/3 of the cell's energy expenditure
[Howarth et al., 2012]. In order to maintain the cell membrane potential, cells keep a low
concentration of sodium ions and high levels of potassium ions within the cell
(intracellular). The sodium-potassium pump moves 3 sodium ions out and moves 2
potassium ions in, thus in total removing one positive charge carrier from the intracellular
space. The action of the sodium-potassium pump is not the only mechanism responsible
for the generation of the resting membrane potential. Also, the selective permeability of the
cell's plasma membrane for the different ions plays an important role. All mechanisms
involved are explained in the main article on generation of the resting membrane potential.
Export of sodium from the cell provides the driving force for several secondary active
transporters membrane transport proteins, which import glucose, amino acids, and other
nutrients into the cell by use of the sodium gradient.
Another important task of the Na+-K+ pump is to provide a Na+ gradient that is used
by certain carrier processes. In the gut, for example, sodium is transported out of the
reabsorbing cell on the blood (interstitial fluid) side via the Na+-K+ pump, whereas, on the
reabsorbing (luminal) side, the Na+-glucose symporter uses the created Na+ gradient as a
source of energy to import both Na+ and glucose, which is far more efficient than simple
diffusion. Similar processes are located in the renal tubular system. Failure of the Na+-K+
pump can result in swelling of the cell. A cell's osmolarity is the sum of the concentrations
of the various ion species and many proteins and other organic compounds inside the cell.
When this is higher than the osmolarity outside of the cell, water flows into the cell
91 through osmosis. This can cause the cell to swell up and lyse. The Na+-K+pump helps to
maintain the right concentrations of ions. Furthermore, when the cell begins to swell, this
automatically activates the Na+-K+pump.
In addition to the classical ion transporting, this membrane protein can also relay
extracellular ouabain-binding signalling into the cell through regulation of protein tyrosine
phosphorylation.
The
downstream
signals
through
ouabain-triggered
protein
phosphorylation events include activation of the mitogen-activated protein kinase (MAPK)
signal cascades, mitochondrial reactive oxygen species (ROS) production, as well as
activation of phospholipase C (PLC) and inositol triphosphate (IP3) receptor (IP3R) in
different intracellular compartments [Yuan et al., 2005].
Protein-protein interactions play a very important role in Na+-K+ pump -mediated
signal transduction. For example, Na+-K+ pump interacts directly with Src, a non-receptor
tyrosine kinase, to form a signaling receptor complex [Tian et al., 2006]. Src kinase is
inhibited by Na+-K+ pump while, upon ouabain binding, the Src kinase domain will be
released and then activated. Based on this scenario, NaKtide, a peptide Src inhibitor
derived from Na+-K+ pump, was developed as a functional ouabain Na+-K+ pump mediated signal transduction [ Li , Cai , Tian , et al., 2009]. Na+-K+ pump also interacts
with ankyrin, IP3R, PI3K, PLC-gamma and cofilin [ Lee et al., 2001].
The Na+-K+ pump has been shown to control and set the intrinsic activity mode of
cerebellar Purkinje neurons [Forrest et al., 2012]. This suggests that the pump might not
simply be a homeostatic, "housekeeping" molecule for ionic gradients; but could be a
computation element in the cerebellum and the brain. Indeed, a mutation in the Na+K+pump causes rapid onset dystonia parkinsonism, which has symptoms to indicate that it
is a pathology of cerebellar computation [Cannon, 2004]. Furthermore, an ouabain block
of Na+-K+pump in the cerebellum of a live mouse results in it displaying ataxia and
dystonia [Calderon, 2011]. The distribution of the Na+-K+pump on myelinated axons, in
human brain, was demonstrated to be along the internodal axolemma, and not within the
nodal axolemma as previously thought [Young, 2008].
92 Mg2+ ATPase:
Magnesium (Mg2+)-ATPase is a mitochondrial membrane-bound enzyme which is
involved in the release of energy from the hydrolysis of ATP. In enzymology, a Mg2+
importing ATPase (EC 3.6.3.2) is an enzyme that catalyzes the chemical reaction.
ATP + H2O + Mg2+out \right left harpoons ADP + phosphate + Mg2+ +in
The 3 substrates of this enzyme are ATP, H2O, and Mg2+, whereas its 3 products
are ADP, phosphate, and Mg2+.This enzyme belongs to the family of hydrolases,
specifically those acting on acid anhydrides to catalyse transmembrane movement of
substances. The systematic name of this enzyme class is ATP phosphohydrolase (Mg2+
importing).The mgt A gene which encodes this enzyme is thought to be regulated by a
magnesium responsive RNA element [Groisman et al., 2006].
Ca2+ATPase:
Ca2+ ATPase is a form of P-ATPase that transfers calcium after a muscle has
contracted. The calcium ATPases are:
(i) Plasma membrane Ca2+ ATPase (PMCA): It is a transport protein in the plasma
membrane of cells that serves to remove calcium (Ca2+) from the cell. It is vital for
regulating the amount of Ca2+ within cells [Jensen et al., 2004]. In fact, the PMCA is
involved in removing Ca2+ from all eukaryotic cells [Strehler et al., 2001]. There is a very
large transmembrane electrochemical gradient of Ca2+ driving the entry of the ion into
cells, yet it is very important for cells to maintain low concentrations of Ca2+ for proper
cell signalling; thus it is necessary for the cell to employ ion pumps to remove the Ca2+
The PMCA and the sodium calcium exchanger (NCX) are together the main regulators of
intracellular Ca2+ concentrations. Since it transports Ca2+ into the extracellular space, the
PMCA is also an important regulator of the calcium concentration in the extracellular
space [Talarico, 2005]. The PMCA belongs to a family of P-type primary ion transport
ATPases that form an aspartyl phosphate intermediate. The PMCA is expressed in a
variety of tissues, including the brain. [Jensen et al., 2006]
93 (ii) Sarcoplasmic reticulum Ca2+ ATPase (SERCA): SERCA resides in the sarcoplasmic
reticulum (SR) within muscle cells. It is a Ca2+ ATPase that transfers Ca2+ from the cytosol
of the cell to the lumen of the SR at the expense of ATP hydrolysis during muscle
relaxation. Sarcoplasmic reticular Ca2+ binding proteins also contribute to decreased
intracellular Ca2+ levels. Recent studies have identified calsequestrin and calreticulin as
sarcoplasmic reticular Ca2+ binding proteins in smooth muscle.
RESULTS:
The levels of various parameters related to energy metabolism, viz., activity of
enzymes (Na+ /K+ -ATPase, Mg2+ -ATPase and Ca2+ -ATPase were estimated in different
regions of brain in control and experimental groups of rats. The enzyme activity levels of
Na+ /K+, Mg2+ and Ca2+ -ATPases in selected brain regions of control albino rats as found
to be highest in Cerebral Cortex (CC), Hippocampus (Hc), Ponsmedulla (Pm) &
Cerebellum (Cb).
Na+/K+ -ATPase: CC
Mg2+ -ATPase:
>
>
(27.47)
(22.72)
CC
Hc
>
(30.94)
Ca2+ -ATPase:
Hc
CC
(17.26)
Hc
(16.75)
>
Cb
(22.54)
>
(25.37)
>
Pm
Pm
(18.37)
>
(23.53)
>
Pm
(14.52)
Cb
(22.25)
>
Cb
(13.44)
When compared to the control rats, the Na+ /K+, Mg2+ and Ca2+ -ATPases in orally
administered EAE treated rats were elevated significantly in all brain regions at selected
time intervals. The percentage of elevation was increased from 20th day to 60th day and
the maximum percentage was noticed in the following order:
94 Na+/K+ -ATPase
(33.86%)
Mg2+ -ATPase:
(23.14%)
Ca2+ -ATPase:
(28.63%)
CC
>
Hc
(26.22%)
Pm
>
> Cb
>
(18.75%)
CC
Cb
>
CC
(24.96%)
Pm
(25.99%)
(22.66%) (18.37%)
Pm
>
>
Hc
(17.26%)
>
Hc
(20.56%)
>
Cb
(14.28%)
On observing the order of four regions of rat brain, it was clear that elevation of
Na+ /K+-ATPase was more in Cerebral cortex (CC) and less in Ponsmedulla (Pm), whereas
Mg2+ -ATPase were recorded higher in Ponsmedulla (Pm) and lower in Hippocampus
(HC) and in case of Ca2+ ATPase, it was observed higher in Ponsmedulla (PM) and lower
in Cerebellum (Cb) regions.
Contrary to the EAE treated rats, the Na+/K+ , Mg2+ and Ca2+ -ATPases in DGalactose injected rats were inhibited significantly in all regions of rat brain at all time
periods. The percentage of inhibition was continuously increased from 20th day to 60th
day and the maximum percentage of inhibition was recorded in the following order:
Na+/K+ -ATPase:
(-33.40%)
Mg2+ -ATPase:
Pm
>
(-33.17%)
Cb
Cb
>
(-30.33%)
>
Hc
(-40.53%) (-37.93%)
Ca2+ -ATPase:
CC
Cb
>
Pm
(-24.96%)
CC
Hc
>
(-38.64%) (-37.85%) (-33.82%) (-32.79%)
95 Hc
(-28.78%)
(-30.97%)
>
>
>
>
CC
Pm
From the above sequence, it was revealed that in AD induced rats, higher percent
change of inhibition in Na+/K+ ATPases was noticed in ponsmedulla and lower in
Hippocampus (HC). With regard to Mg2+ ATPases, highest inhibition was found in
Cerebellum (Cb) and Cerebral Cortex (CC) whereas in case of Ca2+ ATPases, higher
inhibition was recorded in Cerebellum and lower in Ponsmedulla regions of rat brain.
The i.p. injected D-Galactose rats treated simultaneously with EAE showed a
significant inhibition in Na+/K+, Mg2+ and Ca2+ -ATPases of different brain regions from
20th day to 60th day, but the maximum percentage of inhibition was recorded in the
following order:
Na+/K+-ATPase:
CC
>
Cb
(-18.04%) (-17.77%)
Mg2+ -ATPase:
Cb
Cb
Pm
>
Hc
(-16.35%) (-7.16%)
>
Hc
(-17.97%) (-12.32%)
Ca2+ -ATPase:
>
>
Pm
>
CC
(-10.97%) (-6.84%)
> Hc
>
CC
>
Pm
(-18.73%) (-17.16%) (-17.15%) (-14.79%)
On comparing the maximum percent changes of four regions, the enzyme activity
levels of Na+/K+ was higher in cerebral cortex(CC), and lower in Hippocampus(Hc). Mg2+
ATPases were more in cerebellum and least in cerebral cortex. In case of Ca2+ -ATPases,
the enzyme activity levels was more in Cerebellum (Cb) and least in Pons medulla regions
of rat brain.
96 DISCUSSION:
The present study demonstrates the changes that occur in the enzymes connected
with enzyme metabolism and membrane transport functions, viz, Na+/K+ , Mg2+ and Ca2+ ATPases in different brain regions of control and experimental groups of rats at selected
time intervals. The results clearly indicate that oral administration of Evolvulus alsinoides
extract (EAE) significantly elevated the levels of Na+/K+, Mg2+ and Ca2+ -ATPases activity
whereas the i.p. administration of D-Galactose inhibited the ATPase activity in all brain
regions of rat, which could be reverted the changes to the normal level by the consecutive
treatment with EAE up to 60 days continuously.
When compared to control, EAE treated rats showed higher levels of Na+/k+
ATPases and also the concentration increased from 20th day to 60th day. The acetylcholine
released from the nerve terminals attaches to the muscle endplate membrane and activities
the sodium and potassium channels of this area, increasing the rate of diffusion of both
sodium and potassium [James and Breazile, 1983]. As the ACh levels increased from 20th
day to 60th day, the Na+, K+ levels were also increased from 20th day to 60th day.
Potassium is accumulated within human cells by the action of the Na+,K+ ATPase
pump and it is an activator of some enzymes, in particular co-enzyme for normal growth
and muscle function. It helps in protein and carbohydrate metabolism. It is the principle
cation of the intracellular fluid, but it is also a very important constituent of the
extracellular fluid because it influences muscle activity particularly that of the cardiac
muscle. Potassium deficiency causes nervous disorder, diabetes and poor muscular control
resulting in paralysis. The sodium and potassium ion-stimulated adenosine triphosphatase
(Na, K-ATPase) is a membrane-embedded enzyme responsible for the active transport of
Na+ and K+ ions in most animal cells. The enzyme is present in high concentrations in
neurons, where it maintains the gradients of Na+ and K+ necessary for nerve impulse
conduction, and in glial cells, where ion gradients are used for K+ buffering and
neurotransmitter uptake [Stahl, 1986]. In addition, the Na+, K+ ATPase has the potential to
play a role in neuronal signaling: it may modulate synaptic transmission by
97 hyperpolarizing nerve membranes, and it may indirectly regulate the Na+ dependent
carriers for neurotransmitters. Na+, K+ - stimulated ATPase (E.C. 3.6.1.3.) is known to be
involved in the maintenance of sodium and potassium gradients across plasma membranes
at the expense of ATP hydrolysis, with very high activity in electrically excitable tissues.
The activity of this enzyme has been found to increase in the developing rat brain and to
decrease during aging [de Sousa et al., 1978]. The Na+/K+-ATPase maintains the
electrochemical gradients across the plasma membrane, essential for e.g. signaling,
secondary active transport, glutamate re-uptake and neuron excitability in animal cells. The
Na+, K+ ATPase is known to be broadly expressed, exhibit high expression in the
neuromuscular system and a major consumer of cellular energy. Na+/K+-ATPase mutations
are associated with neurological disorders, where mutations in the Na+/K+-ATPase α2 and
α3 isoforms cause Familial hemiplegic migraine type 2 (FHM2) and Rapid-onset dystoniaparkinsonism (RDP)/Alternating hemiplegic childhood (AHC), respectively [Doqanli et
al., 2013]. A decrease in this enzyme activity has been reported to take place in a number
of disorders like neurodegenerative and psychiatric disorders, coronary artery disease and
stroke, syndrome-X, tumors etc [Dhanya et al., 2003].
In a similar way calcium is essential for healthy bones, teeth and blood. It is
required for the absorption of dietary vitamin B, for the synthesis of the neurotransmitter
acetylcholine, for the activation of enzymes such as the pancreatic lipase. It helps to
regulate the activity of skeletal muscle, heart and many other tissues. Deficiency of
calcium causes rickets, osteomalacia and scurvy. The Ca2+ ion is a central signaling
molecule in numerous cellular functions including apoptosis, energy production, gene
regulation, cell proliferation, membrane excitability, synaptic transmission and plasticity.
The Ca2+ ‘dysregulation’ hypothesis of brain aging and Alzheimer’s disease
formulated in the 1980s was based on discrete observations of alterations in processes that
are regulated by Ca2+ [Khachaturian, 1989]. Different mechanisms for Ca2+ dysregulation
contribute to changes in cell excitability [Disterhoft and Oh, 2006] and synaptic plasticity
[Foster, 2007] in the hippocampus. Conversely, in other regions of the nervous system,
changes in Ca2+ regulation may represent compensation to delay physiological aging
98 [Buchholz et al., 2007; Murchison and Griffith, 2007], suggesting that cell specific
differences in the expression of Ca2+ regulating mechanisms may contribute to regional
differences in the rate of brain aging. Similarly, cell specific differences in Ca2+ regulating
mechanisms may interact with neurodegenerative disorders to determine the pattern of cell
death within the brain [LaFerla, 2002; Mattson, 2007; Chan et al., 2009; Naidoo, 2009].
The plasma membrane Ca2+ ATPase is primarily responsible for maintaining rest
[Ca2+], while the Na+/Ca2+ exchanger provides a mechanism for rapidly removing
Ca2+following stimulation but can reverse direction with membrane depolarization. Aβ
markedly impairs Na+/K+-ATPase activity in cultured rat hippocampal neurons and
compromises both the Na+/K+-ATPase and Ca2+ -ATPase in synaptosomes from
postmortem human hippocampus. Impairment of these ion-motive ATPases appears to be a
key step in the cell death process [Robert et al., 1995]. It has been described that Ab
oligomers increase intracellular calcium [Deshpande et al., 2006]. In the present study, it
was showed that Ab in the presence of AChE produces Ab oligomers faster than Ab
aggregates incubated without the enzyme, an effect that could explain why the alterations
triggered by Ab-AChE complexes were stronger at Ca2+ homeostasis level.
It has been described that Ab oligomers/fibrils induce intracellular calcium
deregulation that leads to apoptosis through mitochondria dysfunction, whether by direct
interaction with isolated mitochondria or by indirect association with the neuronal
membrane [Abramov et al., 2004; Casley et al., 2002; Kim et al., 2002; LaFerla et al.,
2002]. One of the earliest effect of Aβ-AChE complexes is an increase in intracellular
calcium, which leads to the loss of the mitochondrial membrane potential, this being in
agreement with the notion that calcium homeostasis and mitochondrial function are the
main targets of these complexes.
Mounting evidence indicates that Ca2+ channels in the membrane contribute to the
specificity of cell loss and the progression of Parkinson’s and Alzheimer’s disease. For
example, in the substantia nigra, a high level of Cav1.3 L-type channel activity contributes
to the discharge pattern of dopamine pacemaker neurons. This activity results in a large
99 Ca2+ influx which is buffered by intracellular stores. However, in Parkinson’s disease,
disruption of this buffering process due to genetic or environmental stress results in toxic
levels of Ca2+ leading to cell death [Chan et al., 2009]. Furthermore, with advanced age,
these neurons increase their reliance on L-channel activity to regulate pacemaker activity,
increasing their liability [Chan et al., 2007]. Thus, the activity of the L-channel provides
the specificity for cell loss in Parkinson’s disease. Temporal lobe regions, including the
hippocampus, exhibit marked cell loss associated with Alzheimer’s disease. The beta
amyloid protein of Alzheimer’s disease, increases cytosolic Ca2+, impairs synaptic
plasticity, and increases cell death through an Lchannel- dependent mechanism [Freir and
Herron, 2003; Fu et al., 2006; Lopez et al., 2008]. Polymorphism of a recently identified
Ca2+ channel has been linked to late-onset Alzheimer’s disease [Dreses Werringloer et
al., 2008]. Interestingly, this gene is predominantly expressed in brain regions, such as the
hippocampus, which exhibit early and profound cell loss. Together, the results indicate that
Ca2+ channels provide a point of cross talk between age-related Ca2+ dysregulation and
signaling in neurodegenerative diseases resulting in selectivity of cell loss.
Reports of age-related changes in the buffering function of the ER and
mitochondria are highly variable across different cell types and brain regions [Brown et
al., 2004]. For example, studies of the peripheral nervous system indicate that buffering of
cytosolic Ca2+ by the ER is decreased during aging [Tsai et al., 1998] with no change in
mitochondrial function [Buchholz et al., 2007]. In contrast, ER-mediated Ca2+ buffering
may be intact [Burnett et al., 1990; Pottorf et al., 2001] and mitochondrial buffering may
be altered at synapses in the central nervous system [Toescu et al., 2000 Murchison et al.,
2004]. Mitochondria from aged animals show structural alterations to mitochondrial DNA
[Cortopassi and Arnheim, 1990; Toescu et al., 2000] and to the mitochondrial membrane
[Kwong and Sohal, 2000], which could contribute to a net decrease in the Ca2+ buffering
capacity during senescence. More important is decreased Ca2+ uptake capacity of aged
mitochondria, which arises as a direct consequence of the decreased electrochemical
gradient across the mitochondrial membrane [Xiong et al., 2004]. Mitochondrial
depolarization may increase the threshold level of Ca2+ needed to initiate mitochondrial
uptake. As such, an age-dependent delay in Ca2+ sequestration or recovery would become
100 apparent under conditions of a large rise in intracellular Ca2+ [Xiong et al., 2002, 2004;
Murchison et al., 2004]. Finally, mitochondria provide a source for oxidative stress and
regional variability in oxidative stress [Dubey et al., 1996; Rebrin et al., 2007] and
mitochondrial damage has been reported [Corral-Debrinski et al., 1992; Filburn et al.,
1996], which appear to correspond to regions that are vulnerable to neurodegenerative
disease.
Daily administration of Convulvlus pluricaulis (CP) (150 mg/kg) for 3 months
along with aluminium chloride (50 mg/kg) decreased the elevated enzymatic activity of
acetylcholine esterase and also inhibited the decline in Na+/K+ATPase activity which
resulted from aluminium intake [Syed et al., 2009]. As C. pluricaulis and E.alsinoides
belongs to the same family convolvulacae, it shares the function where it maintains the
Na+/K+ATPase concentrations. It was proved by the study [Gomathi et al., 2014] investigated that the possible therapeutic effects of the whole plant ethanolic extract of
Evolvulus alsinoides on oral glucose tolerance test and the membrane bound enzyme
activity in streptozotocin induced diabetes rats and the results showed decreased activity of
Na+ K+ ATPase, Mg2+ ATPase and increased activity of calcium ATPase were observed
in streptozotocin induced diabetic rats when compared with control rats. Whereas the
enzyme activities were found to near normal after treatment with ethanolic extract of
Evolvulus alsinoides and glibenclamide treated rats when compared with diabetic rats. Na+
K+-ATPase has been implicated in the development of complications and adaptive
changes in diabetes. In our study the decreased activity of Na+K+ ATPase and increased
activity of calcium ATPase were observed in streptozotocin induced diabetic rats when
compared with normal rats. This is in agreement with the earlier published data of
Mayanil et al., 1982. In experimental diabetes, changes in Na+ K+-ATPase activity have
been reported in the heart, peripheral nerve, kidney and intestine [Gerbi, 1998].
D-Galactose is normally present in the body but when its level increases above the
normal, it gets oxidized into aldehydes and hydrogen peroxide, and Stimulates diabetes
mellitus, induces premature aging with increased serum AGE content and decreases motor
activity. It has also been shown that D-Galactose reduces immune responses, and increases
101 oxidative stress by increasing lipid peroxidation, and decreases antioxidant enzyme
activities and mitochondrial function by inducing degeneration. D-gal has been used to
induce oxidative stress in vivo to mimic the natural aging process in rats and mice [Chen
et al., 2010]. Mitochondria are another important source of ROS production in cells [Raha
and Robinson, 2000]. Mitochondrial ROS production is sensitive to the proton-motive
force across the inner membrane from electron transport. The D-Galactose-induced aging
model could increase the oxidative pressure and inflammation, causing senescence injury.
This senescence-induced model could result in a decline in cognitive function in the brain
and cardiovascular damage. It is widely believed that the longterm injection of DGalactose (D-gal) contributes to the aging progress and slight neuronal damage and
memory deficits which are the prominent changes of AD in the early stage [Chen et al.,
2006]. A decline in body functions is observed during aging partly due to impairments in
various systems in the brain, including cognitive mechanisms. [Seidler et al., 2010] Brain
aging is associated with reduced neurogenesis, increased neurodegeneration, and various
other deficits primarily involving the neurotransmitter systems. Accumulating evidence
suggests
mitochondrial
dysfunction
potently
contributes
to
age-associated
neuropathologies. Reports have described symptoms of brain aging, including impaired
neurogenesis, memory deficits, neurodegeneration, and oxidative damage in rodents as a
result of chronic administration of D-Galactose [Zhang et al., 2005 and Cui et al., 2006].
In the present study D-Galactose is used to build an animal model of ageing in rats. DGalactose impairs brain ATPases by inhibiting the levels of membrane bound enzymes
viz., Na+/K+, Mg2+ and Ca2+ -ATPases. Decrement in levels of ATP, in neurons are
concerned with age related cognitive dysfunction and Alzheimer’s disease.
From the results obtained in the present study, it is inferred that administration of
EAE increased the activities of Na+/K+, Mg2+ and Ca2+ -ATPases and maintained the
concentrations of sodium, potassium, calcium, and magnesium ions in D-Galactose treated
rats brain. In view of the increasing attention towards plants offering non-specific
resistance (adaptogens) towards stress, the improvement in the peripheral stress markers
and scopolamine induced dementia by EAE in one of the study indicates that adaptogenic
and anti-amnesic properties of EAE [Siripurapu, et al., 2005].
102 Ethanolic extracts and water infusion of E. alsinoides were tested for their
antioxidant activity in the 2, 2'- azinobis-3-ethyl-benzothiazoline-6-sulfonic acid radical
cation decolorization assay. Inhibition of lipid peroxidation by plant infusions was carried
out using spontaneous lipid peroxidation of rat brain homogenate, and IC50 values were
determined. The results from the ABTS assay showed that the ethanolic extract of Sida
cordifolia was found to be most potent (IC50 16.07 μg/ml), followed by Evolvulus
alsinoides (IC50 33.39 μg/ml) and Cynodon dactylon (IC50 78.62 μg/ml) [Auddy et al.,
2003].
Evolvulus alsinoides extract reduces lipid peroxidation level and increases the
antioxidant level in experimental rats. It also prevents the pancreas by suppressing the
oxidative stress in associated with diabetes and also help to increase the insulin level by
remodeling the structure of pancreas [Gomathi et al., 2013]. Methanolic extract of
Evolvulus alsinoides herb possessed profound antihypertensive activity in adrenaline
induced hypertensive model [Joshi et al., 2012]. Hydro-alcoholic extract of E. alsinoides
dose dependently prevented STZ induced cognitive impairment by reducing the oxidative
stress, improving cholinergic function and preventing the increase in rho kinase expression
thus suggesting the anti-Alzheimer potential of hydro-alcoholic extract of E. alsinoides
[Mehla et al., 2012]. From the results of the present study, it is concluded that EAE elevated the Na+/K+,
Mg2+ and Ca2+ -ATPase activities in normal rat brain Whereas in D-Galactose treated rats,
it was found that the activities of Na+/K+, Mg2+ and Ca2+ -ATPases inhibited significantly
and EAE could revert these activities to the normal level, indicating that EAE has potential
effect on maintaining ion gradients across biological membranes and thus confer
significant protection to the brain by stabilizing the membrane and there by retaining the
structural and functional integrity of the membrane. 103 Table-19: Changes in Na+, K+-ATPase activity (µmoles of Pi formed/mg protein/h) in
Cerebral Cortex (CC) region of brain from control and experimental groups of
rats on selected time intervals.
Groups
Control
BME
D-Gal
D-Gal
+EAE
20th day
27.477±0.182
34.363±0.213
(+25.06)
21.853±0.178
(-20.46)
21.95±0.259
(-20.09)
40th day
27.954±0.212
36.300±0.148
(+29.85)
19.472±0.170
(-30.34)
22.278±0.154
(-20.30)
60th day
28.142±0.173
37.674±0.277
(+33.86)
18.807±0.157
(-33.17)
23.065±0.196
(-18.04)
Table-20: Changes in Na+, K+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Cerebellum (CB) region of rat brain in experimental groups treated with EAE,
D-Gal and D-Gal & EAE at selected time intervals.
Groups
Control
BME
D-Gal
D-Gal
+EAE
20th day
18.371±0.187
22.903±0.258
(+24.66)
13.41±0.224
(-26.95)
14.088±0.253
(-23.31)
40th day
18.747±0.222
23.508±0.184
(+5.39)
13.320±0.142
(-28.94)
14.292±0.198
(-23.76)
60th day
19.011±0.208
13.243±0.289
(+25.99)
13.243±0.289
(-30.33)
15.632±0.290
(-17.77)
Table-21: Changes in Na+, K+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Hippocampus (HP) region of rat brain in experimental groups treated with
EAE, D-Gal and D-Gal & EAE at selected time intervals.
Groups
Control
BME
D-Gal
D-Gal
+EAE
20th day
22.726±0.297
22.296±0.250
(+24.50)
17.919±0.177
(-21.15)
18.764±0.216
(-17.43)
40th day
23.363±0.232
29.311±0.191
(+25.45)
17.484±0.216
(-25.16)
21.051±0.332
(-9.89)
60th day
23.594±0.146
29.781±0.237
(+26.22)
16.801±0.322
(-28.78)
21.904±0.251
(-7.161)
Values are Mean ± SEM of six observations each from tissues pooled from 6 rats.
Values in parentheses are percent change from control
Values are significantly different from control at p < 0.01
104 Table-22: Changes in Na+, K+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Ponsmedulla (PM) region of rat brain in experimental groups treated with
EAE, D-Gal and D-Gal & EAE at selected time intervals.
Groups
Control
BME
D-Gal
D-Gal
+EAE
20th day
22.544±0.190
26.503±0.226
(+24.66)
15.897±0.167
(-29.48)
17.55±0.277
(-22.14)
40th day
22.869±0.209
26.811±0.274
(+17.23)
15.726±0.227
(-18.75)
18.431±0.293
(-19.38)
60th day
23.06±0.225
27.391±0.330
(+18.75)
15.359±0.226
(-33.40)
19.293±0.267
(-16.35)
Values are Mean ± SEM of six observations each from tissues pooled from 6 rats.
Values in parentheses are percent change from control
Values are significantly different from control at p < 0.01
105 Table -23: One way ANOVA for Na+, K+ -ATPase -20th day.
ANOVA
Sum of
Squares
cerebralcortex Between
Groups
629.956
3
.891
20
630.846
23
403.595
3
1.147
20
Total
404.742
23
Between
Groups
347.272
3
1.084
20
348.357
23
420.164
3
.965
20
421.129
23
Within Groups
Total
hippocampus Between
Groups
Within Groups
cerebellum
Mean
Square
df
Within Groups
Total
ponsmedulla Between
Groups
Within Groups
Total
F
209.985 4.715E3
Sig.
.000
.045
134.532 2.347E3
.000
.057
115.757 2.135E3
.000
.054
140.055 2.904E3
.000
.048
One way ANOVA was performed for four different regions of rat brain for
Na ,K -ATPase activity on 20th day of experiment and the results were showed in the
+
+
table 23. Since Sphericity Assumed test for days is statistically significant with p<0.05. Fvalue were 4.715, 2.347, 2.135, 2.904 for cerebral cortex, hippocampus, cerebellum and
ponsmedulla respectively therefore it was interpreted that the Na+,K+ -ATPase activity is
not same at all time periods and differs significantly at 1% level. Further F-value (p-value
<0.01) for groups is also significant at 1% level under test ‘Between Subjects Effects’.
Further, Dunnet’s Multiple Range Test (DMRT) was conducted for groups, revealed that
the experimental groups (EAE, D-Gal, D-Gal + EAE) differed significantly from that of
control group.
106 Table -24: One way ANOVA for Na+, K+ -ATPase -40th day
ANOVA
Sum of
Squares
cerebralcortex Between
Groups
992.216
3
.600
20
992.816
23
444.179
3
1.240
20
Total
445.419
23
Between
Groups
392.496
3
.717
20
393.212
23
429.998
3
1.283
20
431.281
23
Within Groups
Total
hippocampus Between
Groups
Within Groups
cerebelluum
Mean
Square
df
Within Groups
Total
ponsmedulla Between
Groups
Within Groups
Total
F
330.739 1.103E4
Sig.
.000
.030
148.060 2.389E3
.000
.062
130.832 3.652E3
.000
.036
143.333 2.234E3
.000
.064
One way ANOVA was performed for four different regions of rat brain for
Na+,K+ -ATPase activity on 40th day of experiment and the results were showed in the
table 24. Since Sphericity Assumed test for days is statistically significant with p<0.05. Fvalues were 1.103, 2.389, 3.652, 2.234 for cerebral cortex, hippocampus, cerebellum and
ponsmedulla respectively therefore it was interpreted that the Na+, K+ -ATPase activity is
not same at all time periods and differs significantly at 1% level. Further F-value (p-value
<0.01) for groups is also significant at 1% level under test ‘Between Subjects Effects’.
Further, Dunnet’s Multiple Range Test (DMRT) was conducted for groups, revealed that
the experimental groups (EAE, D-Gal, D-Gal + EAE) differed significantly from that of
control group.
107 Table -25: ONE WAY ANOVA for Na+,K+ -ATPase -60th day.
ANOVA
Sum of
Squares
cerebralcortex Between
Groups
1186.970
3
.852
20
1187.823
23
515.703
3
1.227
20
Total
516.930
23
Between
Groups
388.105
3
1.192
20
389.297
23
477.227
3
1.416
20
478.643
23
Within Groups
Total
hippocampus Between
Groups
Within Groups
cerebellum
Mean
Square
df
Within Groups
Total
ponsmedulla Between
Groups
Within Groups
Total
F
395.657 9.284E3
Sig.
.000
.043
171.901 2.802E3
.000
.061
129.368 2.171E3
.000
.060
159.076 2.247E3
.000
.071
One way ANOVA was performed for four different regions of rat brain for Na+,
K+ -ATPase activity on 60th day of experiment and the results were showed in the table 25.
Since Sphericity Assumed test for days is statistically significant with p<0.05. F-values
were 9.284, 2.802, 2.171, 2.247 for cerebral cortex, hippocampus, cerebellum and
ponsmedulla respectively therefore it was interpreted that the Na+, K+ -ATPase activity is
not same at all time periods and differs significantly at 1% level. Further F-value (p-value
<0.01) for groups is also significant at 1% level under test ‘Between Subjects Effects’.
Further, Dunnet’s Multiple Range Test (DMRT) was conducted for groups, revealed that
the experimental groups (EAE, D-Gal, D-Gal + EAE) differed significantly from that of
control group.
108 Table 26: Changes in Mg2+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Cerebral cortex (CC) region of rat brain in experimental groups treated with
EAE, D-Gal and D-Gal & EAE at selected time intervals.
Groups
Control
BME
D-Gal
D-Gal
+EAE
20th day
30.94±0.083
34.923±0.069
(+12.85)
23.56±0.069
(-23.83)
27.51±0.107
(-11.08)
40th day
31.314±0.057
37.095±0.137
(+18.46)
24.754±0.351
(-20.94)
20.212±0.087
(-9.90)
60th day
31.674±0.099
38.853±0.118
(+22.66)
23.766±0.102
(-24.96)
29.505±0.088
(-6.84)
Table-27: Changes in Mg2+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Cerebellum (CB) region of rat brain in experimental groups treated with
EAE, D-Gal and D-Gal & EAE at selected time intervals.
Groups
Control
BME
D-Gal
D-Gal
+EAE
20th day
22.251±0.078
26.247±0.098
(+17.95)
15.75±0.083
(-29.18)
18.108±0.089
(-18.61)
40th day
23.199±0.100
27.381±0.114
(+18.02)
15.093±0.077
(-34.94)
19.026±0.099
(-17.98)
60th day
24.855±0.067
29.421±0.102
(+18.37)
14.781±0.089
(-40.53)
20.38±0.101
(-17.97)
Table-28: Changes in Mg2+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Hippocampus (HC) region of rat brain in experimental groups treated with
EAE, D-Gal and D-Gal & EAE at selected time intervals.
Groups
Control
BME
D-Gal
D-Gal
+EAE
20th day
40th day
60th day
25.37±0.111
25.764±0.054
26.901±0.008
29.532±0.079
(+16.37)
18.327±0.072
(-27.78)
20.841±0.080
(-17.87)
30.225±0.0677
(+17.31)
17.853±0.092
(-30.70)
21.270±0.064
(-17.44)
31.545±0.061
(+17.26)
16.695±0.106
(-37.93)
23.586±0.056
(-12.32)
Values are Mean ± SEM of six observations each from tissues pooled from 6 rats.
Values in parentheses are percent change from control
Values are significantly different from control at p < 0.01
109 Table-29:
Groups
Control
BME
D-Gal
D-Gal
+EAE
Changes in Mg2+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Ponsmedulla (PM) region of rat brain in experimental groups treated with
EAE, D-Gal and D-Gal & EAE at selected time intervals.
20th day
40th day
60th day
23.538±0.105
24.072±0.138
24.591±0.103
28.617±0.057
(+21.57)
18.291±0.099
(-22.29)
19.60±0.080
(-16.72)
29.736±0.085
(+23.52)
17.499±0.062
(-27.30)
21.210±0.104
(-11.88)
30.282±0.071
(+23.14)
16.974±0.082
(-30.97)
21.89±0.116
(-10.97)
Values are Mean ± SEM of six observations each from tissues pooled from 6 rats.
Values in parentheses are percent change from control
Values are significantly different from control at p < 0.01
110 Table -30: One way ANOVA for Mg2+-ATPase -20th day
ANOVA
Sum of
Squares
cerebralcortex Between
Groups
Mean Square
422.084
3
.141
20
422.225
23
442.421
3
.153
20
Total
442.574
23
Between
Groups
385.731
3
.154
20
385.885
23
387.652
3
.155
20
387.806
23
Within Groups
Total
hippocampus Between
Groups
Within Groups
cerebellum
df
Within Groups
Total
ponsmedulla Between
Groups
Within Groups
Total
F
140.695 1.990E4
Sig.
.000
.007
147.474 1.929E4
.000
.008
128.577 1.671E4
.000
.008
129.217 1.672E4
.000
.008
One way ANOVA was performed for four different regions of rat brain for Mg2+
-ATPase activity activity on 20th day of experiment and the results were showed in the
table 30. Since Sphericity Assumed test for days is statistically significant with p<0.05. Fvalues were 1.990, 1.929, 1.671, 1.672 for cerebral cortex, hippocampus, cerebellum and
ponsmedulla respectively therefore it was interpreted that the Mg2+ -ATPase activity is not
same at all time periods and differs significantly at 1% level. Further F-value (p-value
<0.01) for groups is also significant at 1% level under test ‘Between Subjects Effects’.
Further, Dunnet’s Multiple Range Test (DMRT) was conducted for groups, revealed that
the experimental groups (EAE, D-Gal, D-Gal + EAE) differed significantly from that of
control group.
111 Table -31: One way ANOVA for Mg2+-ATPase -40th day
ANOVA
Sum of
Squares
cerebralcortex Between
Groups
Mean Square
493.818
3
.766
20
494.584
23
521.422
3
.101
20
Total
521.523
23
Between
Groups
505.320
3
.196
20
505.516
23
479.527
3
.207
20
479.734
23
Within Groups
Total
hippocampus Between
Groups
Within Groups
cerebellum
df
Within Groups
Total
ponsmedulla Between
Groups
Within Groups
Total
F
164.606 4.298E3
Sig.
.000
.038
173.807 3.457E4
.000
.005
168.440 1.718E4
.000
.010
159.842 1.548E4
.000
.010
One way ANOVA was performed for four different regions of rat brain for Mg2+ ATPase activity on 40th day of experiment and the results were showed in the table 31.
Since Sphericity Assumed test for days is statistically significant with p<0.05. F-values
were 4.298, 3.457, 1.718, 1.548for cerebral cortex, hippocampus, cerebellum and
ponsmedulla respectively therefore it was interpreted that the Mg2+ -ATPase activity is not
same at all time periods and differs significantly at 1% level. Further F-value (p-value
<0.01) for groups is also significant at 1% level under test ‘Between Subjects Effects’.
Further, Dunnet’s Multiple Range Test (DMRT) was conducted for groups, revealed that
the experimental groups (EAE, D-Gal, D-Gal + EAE) differed significantly from that of
control group
112 Table -32: One way ANOVA for Mg2+-ATPase -60th day.
ANOVA
Sum of
Squares
cerebralcortex Between
Groups
Mean Square
700.077
3
.212
20
700.288
23
702.109
3
.128
20
Total
702.236
23
Between
Groups
704.477
3
.167
20
704.644
23
554.046
3
.182
20
554.228
23
Within Groups
Total
hippocampus Between
Groups
Within Groups
cerebellum
df
Within Groups
Total
ponsmedulla Between
Groups
Within Groups
Total
F
233.359 2.206E4
Sig.
.000
.011
234.036 3.668E4
.000
.006
234.826 2.806E4
.000
.008
184.682 2.033E4
.000
.009
One way ANOVA was performed for four different regions of rat brain for Mg2+ ATPase activity activity on 60th day of experiment and the results were showed in the table
32. Since Sphericity Assumed test for days is statistically significant with p<0.05. F-values
were 2.206, 3.668, 2.806, 2.033for cerebral cortex, hippocampus, cerebellum and
ponsmedulla respectively therefore it was interpreted that the Mg2+ -ATPase activity is not
same at all time periods and differs significantly at 1% level. Further F-value (p-value
<0.01) for groups is also significant at 1% level under test ‘Between Subjects Effects’.
Further, Dunnet’s Multiple Range Test (DMRT) was conducted for groups, revealed that
the experimental groups (EAE, D-Gal, D-Gal + EAE) differed significantly from that of
control group
113 Table-33: Changes in Ca2+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Cerebral cortex (CC) region of rat brain in experimental groups treated with
EAE, D-Gal and D-Gal & EAE at selected time intervals.
Groups
Control
BME
D-Gal
D-Gal
+EAE
20th day
17.265±0.053
21.467±0.055
(+24.34)
11.74±0.058
(-31.98)
13.94±0.114
(-19.23)
40th day
17.668±0.098
21.912±0.061
(+24.02)
11.461±0.060
(-35.13)
13.784±0.063
(-21.98)
60th day
17.961±0.062
22.445±0.055
(+24.96)
11.16±0.070
(-37.85)
14.879±0.069
(-17.15)
Table- 34: Changes in Ca2+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Cerebellum (CB) region of rat brain in experimental groups treated with EAE,
D-Gal and D-Gal & EAE at selected time intervals.
Groups
20th day
40th day
60th day
Control
13.447±0.056
15.274±0.664
(+13.58)
9.687±0.054
(-27.95)
10.144±0.091
(-24.56)
13.934±0.058
15.726±0.046
(+2.843)
9.145±0.069
(-34.365)
11.175±0.071
(-19.798)
14.503±0.083
16.575±0.057
(+14.28)
8.898±0.070
(-38.645)
11.786±0.068
(-18.73)
BME
D-Gal
D-Gal
+EAE
Table-35: Changes in Ca2+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Hippocampus (HC) region of rat brain in experimental groups treated with
EAE, D-Gal and D-Gal & EAE at selected time intervals.
Groups
Control
BME
D-Gal
D-Gal
+EAE
20th day
16.757±0.042
19.92±0.066
(+18.88)
13.262±0.063
(-12.484)
14.665±0.063
(-12.48)
40th day
17.121±0.078
20.309±0.066
(+18.619)
12.040±0.061
(-33.82)
13.998±0.045
(-18.24)
60th day
17.659±0.060
21.290±0.040
(+20.56)
11.685±0.061
(-33.82)
14.627±0.312
(-17.16)
Values are Mean ± SEM of six observations each from tissues pooled from 6 rats.
Values in parentheses are percent change from control
Values are significantly different from control at p < 0.01
114 Table-36:
Groups
Control
BME
D-Gal
D-Gal
+EAE
Changes in Ca2+ -ATPase activity (µmoles of Pi formed/mg protein/h) in
Ponsmedulla (PM) region of rat brain in experimental groups treated with
EAE, D-Gal and D-Gal & EAE at selected time intervals.
20th day
14.526±0.053
18.268±0.075
(+27.75)
11.364±0.055
(-21.768)
12.102±0.076
(-16.68)
40th day
14.736±0.357
18.988±0.053
(+28.85)
10.930±0.043
(-25.82)
12.524±0.073
(-15.01)
60th day
15.44±0.060
19.86±0.559
(+28.63)
10.361±0.100
(-32.79)
13.159±0.045
(-14.79)
Values are Mean ± SEM of six observations each from tissues pooled from 6 rats.
Values in parentheses are percent change from control
Values are significantly different from control at p < 0.01
115 Table -37: One way ANOVA for Ca 2+ -ATPase -20th day
ANOVA
Sum of
Squares
cerebralcortex Between
Groups
322.839
3
.112
20
322.951
23
150.834
3
.073
20
Total
150.907
23
Between
Groups
129.180
3
.095
20
129.274
23
174.152
3
.088
20
174.241
23
Within Groups
Total
hippocampus Between
Groups
Within Groups
cerebellum
Mean
Square
df
Within Groups
Total
ponsmeedulla Between
Groups
Within Groups
Total
F
107.613 1.919E4
Sig.
.000
.006
50.278 1.379E4
.000
.004
43.060 9.096E3
.000
.005
58.051 1.315E4
.000
.004
One way ANOVA was performed for four different regions of rat brain for Ca 2+ ATPase activity activity on 20th day of experiment and the results were showed in the table
37. Since Sphericity Assumed test for days is statistically significant with p<0.05. F-values
were 1.919, 1.379, 9.096, 1.315for cerebral cortex, hippocampus, cerebellum and
ponsmedulla respectively therefore it was interpreted that the Mg2+ -ATPase activity is not
same at all time periods and differs significantly at 1% level. Further F-value (p-value
<0.01) for groups is also significant at 1% level under test ‘Between Subjects Effects’.
Further, Dunnet’s Multiple Range Test (DMRT) was conducted for groups, revealed that
the experimental groups (EAE, D-Gal, D-Gal + EAE) differed significantly from that of
control group.
116 Table -38: One way ANOVA for Ca 2+ -ATPase -40th day
ANOVA
Sum of
Squares
cerebralcortex Between
Groups
Mean Square
378.493
3
.105
20
378.598
23
236.685
3
.074
20
Total
236.759
23
Between
Groups
152.837
3
.078
20
152.915
23
220.047
3
.690
20
220.737
23
Within Groups
Total
hippocampus Between
Groups
Within Groups
cerebellum
df
Within Groups
Total
ponsmedulla Between
Groups
Within Groups
Total
F
126.164 2.394E4
Sig.
.000
.005
78.895 2.132E4
.000
.004
50.946 1.306E4
.000
.004
73.349 2.128E3
.000
.034
One way ANOVA was performed for four different regions of rat brain for Ca 2+ ATPase activity activity on 40th day of experiment and the results were showed in the table
38. Since Sphericity Assumed test for days is statistically significant with p<0.05. F-values
were 2.394, 2.132, 1.306, 2.128 for cerebral cortex, hippocampus, cerebellum and
ponsmedulla respectively therefore it was interpreted that the Mg2+ -ATPase activity is not
same at all time periods and differs significantly at 1% level. Further F-value (p-value
<0.01) for groups is also significant at 1% level under test ‘Between Subjects Effects’.
Further, Dunnet’s Multiple Range Test (DMRT) was conducted for groups, revealed that
the experimental groups (EAE, D-Gal, D-Gal + EAE) differed significantly from that of
control group
117 Table -39: One way ANOVA for Ca 2+ -ATPase -60th day
ANOVA
Sum of
Squares
cerebralcortex Between
Groups
Mean Square
411.360
3
.084
20
411.443
23
305.055
3
.533
20
Total
305.588
23
Between
Groups
199.942
3
.099
20
200.041
23
290.722
3
.095
20
290.818
23
Within Groups
Total
hippocampus Between
Groups
Within Groups
cerebellum
df
Within Groups
Total
ponsmedulla Between
Groups
Within Groups
Total
F
137.120 3.271E4
Sig.
.000
.004
101.685 3.816E3
.000
.027
66.647 1.340E4
.000
.005
96.907 2.035E4
.000
.005
One way ANOVA was performed for four different regions of rat brain for Ca 2+ ATPase activity activity on 60th day of experiment and the results were showed in the table
39. Since Sphericity Assumed test for days is statistically significant with p<0.05. F-values
were 3.271, 3.816, 1.340, 2.035for cerebral cortex, hippocampus, cerebellum and
ponsmedulla respectively therefore it was interpreted that the Mg2+ -ATPase activity is not
same at all time periods and differs significantly at 1% level. Further F-value (p-value
<0.01) for groups is also significant at 1% level under test ‘Between Subjects Effects’.
Further, Dunnet’s Multiple Range Test (DMRT) was conducted for groups, revealed that
the experimental groups (EAE, D-Gal, D-Gal + EAE) differed significantly from that of
control group
118 Figure-5 : Graphical representation of percent changes in the activity of Na+,K+ -ATPase
(invivo) in Cerebral cortex(CC), Cerebellum (CB), Hippocampus (HP),
Ponsmedulla (PM), regions of Experimental groups of Rats treated with
EAE, D-Galactose and D-Galactose + EAE.
Cerebral Cortex
Cerebelllum
40
30
30
20
20
10
10
0
0
‐10
20 th day
40 th day
‐10
60 th day
20 th day
40 th day
60 th day
‐20
‐20
‐30
‐30
‐40
‐40
Hippocampus
Ponsmedulla
EAE
30
30
20
20
10
10
0
D‐GAL
EAE+D‐GAL
0
20 th day
40 th day
60 th day
‐10
‐10
‐20
‐20
‐30
‐30
‐40
‐40
119 20 th day 40 th day 60 th day
Figure-6: Graphical representation of percent changes in the activity of Mg2+ -ATPase
(invivo) in Cerebral cortex(CC), Cerebellum (CB), Hippocampus (HP),
Ponsmedulla (PM), regions of Experimental groups of Rats treated with
EAE, D-Galactose and D-Galactose + EAE.
Cerebral cortex
Cerebellum
30
30
20
20
10
10
0
0
20 th day
40 th day
‐10
60 th day
‐10
20 th day
40 th day
60 th day
‐20
‐30
‐20
‐40
‐30
‐50
Hippocampus
Ponsmedulla
30
30
20
20
10
10
0
‐10
D‐GAL
D‐GAL+EAE
0
20 th day
40 th day
60 th day
20 th day
‐10
‐20
‐20
‐30
‐30
‐40
‐40
‐50
120 EAE
40 th day
60 th day
Figure-7: Graphical representation of percent changes in the activity of Ca2+ -ATPase
(invivo) in Cerebral cortex(CC), Cerebellum (CB), Hippocampus (HP),
Ponsmedulla (PM), regions of Experimental groups of Rats treated with EAE,
D-Galactose and D-Galactose + EAE.
Cerebral Cortex
Cerebellum
20
30
10
20
10
0
0
‐10
‐10
20 th day
40 th day
60 th day
‐20
20 th day
40 th day
‐20
‐30
‐30
‐40
‐40
‐50
‐50
Hippocampus
Ponsmedulla
30
40
20
30
EAE
D‐GAL
20
10
D‐GAL+EAE
10
0
‐10
20 th day
40 th day
0
60 th day
‐10
‐20
‐20
‐30
‐30
‐40
‐40
121 60 th day
20 th day
40 th day
60 th day